Glycolipids and analogues thereof as antigens for NKT cells

ABSTRACT

This invention relates to immunogenic compounds which serve as ligands for NKT (natural killer T) cells and to methods of use thereof in modulating immune responses.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a continuation of U.S. patent application Ser. No.11/735,313, filed Apr. 13, 2007, currently pending, which is acontinuation-in-part of U.S. patent application Ser. No. 11/317,900,filed Dec. 27, 2005, now U.S. Pat. No. 7,534,434, issued May 19, 2009,which claims the benefit of U.S. Provisional Patent Application Ser. No.60/639,408, filed Dec. 28, 2004, each of which is incorporated byreference herein in its entirety.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

This invention was made in whole or in part with government supportunder grant number GM44154, awarded by the National Institute of Health.The government may have certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to immunogenic compounds that serve as ligandsfor natural killer T (NK T) cells and to methods of use thereof inmodulating immune responses.

BACKGROUND OF THE INVENTION

CD1 molecules are a family of highly conserved antigen presentingproteins that are similar in function to classical MHC molecules. Whileclassical MHC molecules present peptides, CD1 proteins bind and displaya variety of lipids and glycolipids to T lymphocytes. The five knownisoforms are classified into two groups, group I (CD1a, CD1b, CD1c, andCD1e in humans) and group II (CD1d in humans and mice) based onsimilarities between nucleotide and amino acid sequences.

A great diversity of lipids and glycolipids has been shown to bindspecifically to each of the four isoforms. The first crystal structureof murine (m) CD1d revealed that it adopts a folded conformation closelyrelated to major histocompatibility complex (MHC) class I proteins. Itfurther revealed that mCD1d could accommodate long lipid tails in twohydrophobic pockets, designated A′ and F′, located in the bindinggroove. Two additional structures of hCD1b and hCD1a confirmed thismodel by demonstrating that CD1, when loaded with antigenic glycolipids,binds the lipid portion in a hydrophobic groove while making availablethe hydrophilic sugar moiety to make contact with the T-cell receptor.

Mammalian and mycobacterial lipids are known to be presented by humanCD1a, CD1b, CD1c and CD1d [Porcelli, S. A. & Modlin, R. L. (1999) Annu.Rev. Immunol. 17, 297-329]. α-Galactosylceramide (α-GalCer), a lipidfound in the marine sponge Agelas mauritianus, has been, to date, themost extensively studied ligand for CD1d. α-GalCer, when bound to CD1d,stimulates rapid Th1 and Th2 cytokine production by Vα14i natural killerT (Vα14i NKT) cells in mice and the human homologue Vα24i NKT cells.However, its physiological significance in mammals remains unclear as itis enigmatic why an α-galactosyl ceramide of marine origin is such apotent agonist. Other known ligands, such as a bacterial phospholipid(PIM₄), a tumor derived ganglioside GD3, a C-linked analog of α-GalCer,α-GalCer analogues with different lipid chain lengths and aphosphoethanolamine, found in human tumor extracts, stimulate onlyrelatively small populations of CD1d-restricted NKT cells.

Natural Killer (NK) cells typically comprise approximately 10 to 15% ofthe mononuclear cell fraction in normal peripheral blood. Historically,NK cells were first identified by their ability to lyse certain tumorcells without prior immunization or activation. NK cells also serve acritical role in cytokine production, which are thought to be involvedin controlling cancer, infection and fetal implantation.

In view of the above, it would be desirable to elucidate additionalglycolipid compounds and/or analogues of known glycolipids, such asα-GalCer, that enhance cell mediated immune responses and that areuseful for the development of glycolipid-containing adjuvants andvaccines and for any immunotherapy designed to stimulate NKT cells.

SUMMARY OF THE INVENTION

The present invention is based in part on the discovery of novelglycolipid analogues that enhance cell mediated immune responses andthat are useful for developing vaccines for treating a variety ofdiseases and disorders requiring immunotherapy and stimulation of NK Tcells.

This invention provides, in one embodiment, a compound represented bythe structure of formula 1:

-   -   wherein, R═COOR₁ or CH₂OR₁;        -   R₁═H or an alkyl group;        -   R₂═H or SO₃ ⁻;        -   R₃═H or OH;        -   R₃′═H or OH;        -   R₄ and R₄′ are independently chosen from        -   (a) H,        -   (b) alkyl;        -   (c) alkenyl; and        -   (d) oxaalkyl;        -   R₅═OH, acetamido or a halogen atom;        -   or a pharmaceutically acceptable salt thereof,        -   wherein if R═CH₂OR₁, R₂═H, R₃ is OH and R₃′ is H, then one            of the following is true:            -   (i) R₅=acetamido, halogen atom or OH in an axial                position;            -   (ii) R₄═H, or unsaturated or saturated alkyl chain                having 9 carbon atoms or fewer; or            -   (iii) R₄′═H, or unsaturated or saturated alkyl chain                having 20 carbon atoms or fewer.

In another embodiment, this invention provides a compound represented bythe structure of formula 2:

-   -   wherein R═COOR₁ or CH₂OR₁;        -   R₁═H or an alkyl group;        -   R₂═H or SO₃ ⁻;        -   R₃═H or OH;        -   R₃′═H or OH; and        -   R₄═H, unsaturated or saturated, alkyl group; and        -   R₄′═H, unsaturated or saturated, alkyl group;    -   or a pharmaceutically acceptable salt thereof,        -   wherein if R═CH₂OR₁, R₂═H, R₃ is OH and R₃′ is H, then R₄═H,            unsaturated or saturated, alkyl chain having 9 carbon atoms            or fewer, or R₄′═H, unsaturated or saturated, alkyl chain            having 20 carbon atoms or fewer.

In another embodiment, this invention provides a compound represented bythe structure of formula 3:

-   -   wherein, R═COOR₁ or CH₂OR₁;        -   R₁═H or an alkyl group;        -   R₂═SO₃ ⁻; and        -   n=integer;    -   or a pharmaceutically acceptable salt thereof.

In another embodiment, this invention provides a compound represented bythe structure of formula 4:

-   -   or a pharmaceutically acceptable salt thereof.

In another embodiment, the salt may be, inter alia, a sodium salt.

In another embodiment, this invention provides a compound represented bythe structure of formula 5:

In another embodiment, this invention provides a compound represented bythe structure of formula 6:

In another embodiment, this invention provides a compound represented bythe structure of formula 7:

In another embodiment, this invention provides a compound represented bythe structure of formula 8:

In one embodiment, this invention provides a compound represented by thestructure of formula 9:

-   -   wherein, R═COOR₁ or CH₂OR₁        -   R₁═H or an alkyl group;        -   R₂═H or SO₃ ⁻;        -   R₃═OH;        -   R₃′═H or OH; and        -   R₄═H, unsaturated or saturated, alkyl group; and        -   R₄′═H, unsaturated or saturated, alkyl group;            -   or a pharmaceutically acceptable salt thereof,        -   wherein if R═CH₂OR₁, R₂═H, R₃ is OH and R₃′ is H, then R₄═H,            unsaturated or saturated, alkyl chain having 9 carbon atoms            or fewer, or R₄′═H, unsaturated or saturated, alkyl chain            having 20 carbon atoms or fewer.

In another embodiment, this invention provides a compound represented bythe structure of formula 10:

or a pharmaceutically acceptable salt thereof. In another embodiment,the salt may be, inter alia, a sodium salt.

This invention provides, in one embodiment, a compound represented bythe structure of formula 11:

-   -   wherein, R═COOR₁ or CH₂OR₁;        -   R₁═H or an alkyl group;        -   R₂═H or SO₃ ⁻;        -   R₃═H or OH;        -   R₄═H, unsaturated or saturated, alkyl group;        -   R₅═OH, acetamido or a halogen atom; and        -   R₆═X-A        -   A=        -   dialkyl phenyl;

-   -   X=alkyl, alkenyl, alkoxy, thioalkoxy, substituted furan, or        unsubstituted furan;    -   Y═N or C    -   R7=halogen, H, phenyl, alkyl, alkoxy, nitro or CF3; and    -   R8=methyl or H;    -   or a pharmaceutically acceptable salt thereof.

In another embodiment, this invention provides a compound represented bythe structure of formula 12:

-   -   or a pharmaceutically acceptable salt thereof.

In another embodiment, this invention provides a compound represented bythe structure of formula 13:

-   -   or a pharmaceutically acceptable salt thereof.

In another embodiment, this invention provides a compound represented bythe structure of formula 14:

-   -   or a pharmaceutically acceptable salt thereof.

In another embodiment, the salt may be, inter alia, a sodium salt.

In another embodiment, this invention provides a compound represented bythe structure of formula 15:

In another embodiment, this invention provides a compound represented bythe structure of formula 16:

In still another embodiment, this invention provides a compoundrepresented by the structure of formula 17:

-   -   wherein    -   R₄ and R₆ are independently selected from:    -   (a) alkyl,    -   (b) alkenyl,    -   (c) alkyl terminating in aryl, substituted aryl, heteroaryl or        substituted heteroaryl; and    -   (d) alkenyl terminating in aryl, substituted aryl, heteroaryl or        substituted heteroaryl.

In certain embodiments of compounds of formula 17,

-   -   R₆ is

-   -   X is an alkyl chain; and    -   R₇ is chosen from halogen, H, phenyl, alkyl, alkoxy, nitro, and        CF₃.

In other embodiments of compounds of formula 17,

-   -   R₄ is

-   -   X is an alkyl chain; and    -   R₇ is chosen from halogen, H, phenyl, alkyl, alkoxy, nitro, and        CF₃.

In one embodiment, any one of the compounds of the invention may be aligand for an NKT (natural killer T) cell. In another embodiment, theligand may be in a complex with a CD1 molecule. In another embodiment,the CD1 molecule is a CD1d molecule. In another embodiment, the ligandstimulates NKT cells, which express a CD161+NK marker as well as aninvariant T cell antigen receptor (TCR) on the surface thereof.

In another embodiment, the invention provides a composition or vaccineincluding, inter alia, any one of the compounds of the invention. Inanother embodiment, the composition or vaccine may include, inter alia,at least one cell population. In another embodiment, the cell populationmay include, inter alia, NKT cells, antigen presenting cells, or acombination thereof.

In another embodiment, the invention provides a method for stimulatingNKT cells, the method includes, inter alia, contacting an NKT cell withany one of the compounds of the invention.

In another embodiment, the invention provides a cell population obtainedby any one of the methods of the invention.

In another embodiment, the invention provides a method for stimulating,inhibiting, suppressing or modulating an immune response in a subject,the method includes, inter alia, the step of contacting an NKT cell inthe subject with any one of the compounds of the invention.

In another embodiment, the compound according to the invention may be ina complex with a CD1 molecule. In another embodiment, the CD1 moleculemay be CD1d. In another embodiment, the complex may be displayed on adendritic cell. In another embodiment, the complex may be displayed onany antigen presenting cell.

In one embodiment of the invention, the NKT cells secrete a cytokine. Inanother embodiment the NKT cell may be a Vα24iNKT cell in humans. Inanother embodiment the NKT cell may be a Vα14i NKT cell in mice.

In one embodiment of the invention, the subject may beimmunocompromised. In another embodiment, the subject is infected. Inanother embodiment, the subject is infected with HIV. In anotherembodiment, the subject is infected with mycobacteria. In anotherembodiment, the subject is infected with malaria. In another embodiment,the subject is infected with HIV, mycobacteria, or malaria.

In one embodiment of the invention, the subject is afflicted withcancer. In one embodiment of the invention, the subject is at anelevated risk for cancer. In one embodiment of the invention, thesubject has precancerous precursors.

In one embodiment of the invention, the immune response is biased towardTh1 or Th2. In another embodiment, the subject suffers from, or is at anelevated risk for an autoimmune disease. In another embodiment, thebiasing of the immune response results in the suppression, inhibition orabrogation of the autoimmune disease. In another embodiment, the subjecthas an inappropriate or undesirable immune response. In anotherembodiment, the response is inflammatory. In another embodiment, theinappropriate or undesirable response exacerbates an infection, diseaseor symptom in the subject.

In another embodiment, the invention provides an adjuvant including,inter alia, any one of the compounds according to the invention.

In another embodiment, the invention provides a method of enhancingimmunogenicity of a compound, composition, or vaccine in a subject, themethod includes, inter alia, administering to the subject a compound,composition or vaccine further comprising an adjuvant of according tothe invention, wherein the adjuvant enhances the immunogenicity of thecompound, composition or vaccine.

In another embodiment, the invention provides a method of stimulating orenhancing cytokine production in a subject, the method includes, interalia, administering to the subject any one of the compounds of theinvention, whereby an NKT cell in the subject secretes a cytokinefollowing contact with the compound. In another embodiment, the cytokinemay be interferon-γ or Interleukin-4.

Furthermore, in one embodiment, the invention provides a process for thepreparation of a compound represented by the structure of formula (4)

-   -   or a pharmaceutically salt thereof, the process includes, inter        alia, the step of:        -   removing the benzyldiene protecting group and hydrogenating            of the compound represented by the structure of formula            (4a),

or a salt thereof, wherein PG is a hydroxy protecting group. In anotherembodiment, the hydroxy protecting group may be benzyl.

In one embodiment of the invention, the compound of formula (4a) may beobtained by a process that includes, inter alia, the step of:

-   -   conducting a selective sulfation of the 3″ OH of the galactose        moiety of the compound represented by the structure of formula        (4b):

wherein PG is a hydroxy protecting group and R is H. In anotherembodiment, the hydroxy protecting group may be benzyl.

In one embodiment of the invention, the compound of formula (4b) whereinR is H, may be obtained by a process including, inter alia, the step ofremoving the levulinyl protecting group of a compound of formula (4b)wherein R is levulinyl, thereby obtaining a compound of formula (4b)wherein R is H.

In one embodiment of the invention, the compound of formula (4b) whereinR is levulinyl may be obtained by a process including, inter alia, thestep of:

-   -   reacting a compound represented by the structure of formula        (4c):

wherein R is H or levulinyl with hexacosanoic acid, thereby obtainingthe compound of formula (4b) wherein R is levulinyl.

In one embodiment of the invention, the compound of formula (4c),wherein R is H or levulinyl, may be obtained by a process including,inter alia, the step of:

-   -   reducing the azide group of a compound represented by the        structure of formula (4d):

-   -   wherein R is levulinyl, thereby obtaining a compound of formula        (4c) wherein R is H or levulinyl.

In one embodiment of the invention, the compound of formula (4d) whereinR is levulinyl, may be obtained by a process including, inter alia, thestep of:

-   -   reacting a compound represented by the structure of formula (4e)

-   -   wherein PG is a hydroxy protecting group, LG is a leaving group        and R is levulinyl,    -   with a compound represented by the structure of formula (4f)

-   -   wherein PG is a hydroxy protecting group,    -   to form an alpha glycosidic bond, thereby obtaining the compound        of formula (4d) wherein R is levulinyl. In another embodiment,        the leaving group may be, inter alia,

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of formula (10)

-   -   or a pharmaceutically salt thereof, including, inter alia, the        step of:    -   conducting a selective sulfation of the 3″ OH of the galactose        moiety of the compound represented by the structure of formula        (10a):

-   -   In another embodiment, the sulfation may be conducted in the        presence of Bu₂SnO.

In one embodiment of the invention, the compound of formula (10a) may beobtained by the process including, inter alia, the step of:

-   -   removing the hydroxy protecting groups and hydrogenating the        compound represented by the structure of formula (10b):

wherein PG and PG₁ are hydroxy protecting groups, thereby obtaining thecompound of formula (10a). In another embodiment, the PG may be, interalia, benzyl. In another embodiment, the PG1 may be, inter alia,benzoyl. In one embodiment of the invention, the compound of formula(10b) may be obtained by a process including, inter alia, the step of:

-   -   reacting a compound represented by the structure of formula        (10c):

-   -   wherein PG is a hydroxy protecting group,    -   with a compound represented by the structure of formula (10d):

wherein PG₁ is a hydroxy protecting group and LG is a leaving group,thereby obtaining the compound of formula (10b). In another embodiment,the leaving group may be, inter alia,

In one embodiment of the invention, the compound of formula (10c) may beobtained by a process including, inter alia, the steps of:

-   -   reducing the azide of a compound represented by the structure of        formula (10e):

-   -   wherein PG and PG₂ are hydroxy protecting groups;    -   reacting the resulting amine with hexacosanoic acid; and        removing the hydroxy protecting group PG₂, thereby obtaining the        compound of formula (10c). In another embodiment, the PG₂ may        be, inter alia, TIPS.

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of formula (18):

-   -   or a pharmaceutically salt thereof, including, inter alia, the        step of:    -   conducting a selective sulfation of the 3″ OH of the galactose        moiety of the compound represented by the structure of formula        (18a):

thereby obtaining the a compound represented by the structure of formula(18). In another embodiment, the sulfation may be conducted in thepresence of Bu₂SnO.

In one embodiment of the invention, the compound of formula (18a) may beobtained by the process including, inter alia, the step of:

-   -   removing the hydroxy protecting groups of the compound        represented by the structure of formula (18b):

wherein PG and PG₁ are hydroxy protecting groups, thereby obtaining thecompound of formula (18a). In another embodiment, PG may be, inter alia,benzoyl. In another embodiment, PG₁ may be, inter alia, benzoyl. In oneembodiment of the invention, the compound of formula (18b) may beobtained by a process including, inter alia, the step of:

-   -   deprotecting the amine of a compound represented by the        structure of formula (18c):

-   -   wherein PG and PG₁ are hydroxy protecting groups, and    -   PG₃ is an amino protecting group,    -   and reacting with nervonic acid, thereby obtaining the compound        of formula (18b). In another embodiment, the amino protecting        group may be, inter alia, tBoc.

Furthermore, in one embodiment, the invention provides a process for thepreparation of a compound represented by the structure of formula (13)

-   -   or a pharmaceutically salt thereof, wherein R is CH₂OH and R₂ is        H, the process including, inter alia, the step of:        -   removing the benzyldiol protecting group and hydrogenating            of the compound represented by the structure of formula            (13a),

or a salt thereof, wherein PG is a hydroxy protecting group, R₂ is H. Inanother embodiment, the hydroxy protecting group may be benzyl.

In one embodiment of the invention, the compound of formula (13a)wherein R₂O is SO₃ ⁻, may be obtained by a process including, interalia, the step of conducting a selective sulfation of the 3″ OH of thegalactose moiety of the compound represented by the structure of formula(13a).

In one embodiment of the invention, the compound of formula (13a)wherein R₂ is H, may be obtained by a process including, inter alia, thestep of removing the levulinyl protecting group of a compound of formula(13b) wherein R₂ is levulinyl, thereby obtaining a compound of formula(13a) wherein R₂ is H.

In one embodiment of the invention, the compound of formula (13b)wherein R₂ is levulinyl may be obtained by a process including, interalia, the step of:

-   -   reacting a compound represented by the structure of formula        (13c):

wherein R₂ is levulinyl with an acid form of R₆, thereby obtaining thecompound of formula (13b) wherein R₂ is levulinyl.

In one embodiment of the invention, the compound of formula (13c),wherein R₂ is levulinyl, may be obtained by a process including, interalia, the step of:

-   -   reducing the azide group of a compound represented by the        structure of formula (13d):

-   -   wherein R₂ is levulinyl, thereby obtaining a compound of formula        (13c).

In one embodiment of the invention, the compound of formula (13d)wherein R₂ is levulinyl, may be obtained by a process including, interalia, the step of:

-   -   reacting a compound represented by the structure of formula        (13e)

-   -   wherein PG is a hydroxy protecting group, LG is a leaving group        and R₂ is levulinyl,    -   with a compound represented by the structure of formula (13f)

-   -   wherein PG is a hydroxy protecting group,    -   to form an alpha glycosidic bond, thereby obtaining the compound        of formula (13d) wherein R₂ is levulinyl. In another embodiment,        the leaving group may be, inter alia,

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of formula (14)

-   -   or a pharmaceutically salt thereof, including, inter alia, the        step of:    -   conducting a selective sulfation of the 3″ OH of the galactose        moiety of the compound represented by the structure of formula        (13):

-   -   Wherein R₂ is H and R is CH₂OH.    -   In another embodiment, the sulfation may be conducted in the        presence of Bu₂SnO.

In one embodiment of the invention, the compound of formula (13) may beobtained by the process including, inter alia, the step of:

-   -   removing the hydroxy protecting groups and hydrogenating the        compound represented by the structure of formula (13g):

wherein PG and PG₁ are hydroxy protecting groups, thereby obtaining thecompound of formula (13), wherein R₂ is H and R is CH₂OH. In anotherembodiment, the PG may be, inter alia, benzyl. In another embodiment,the PG1 may be, inter alia, benzoyl. In one embodiment of the invention,the compound of formula (13g) may be obtained by a process including,inter alia, the step of:

-   -   reacting a compound represented by the structure of formula        (13h):

-   -   wherein PG is a hydroxy protecting group,    -   with a compound represented by the structure of formula (13i):

wherein PG₁ is a hydroxy protecting group and LG is a leaving group,thereby obtaining the compound of formula (13g). In another embodiment,the leaving group may be, inter alia,

In one embodiment of the invention, the compound of formula (13h) may beobtained by a process comprising the steps of:

-   -   reducing the azide of a compound represented by the structure of        formula (13j):

-   -   wherein PG and PG₂ are hydroxy protecting groups;    -   reacting the resulting amine with an acid form of R₆; and        removing the hydroxy protecting group PG₂, thereby obtaining the        compound of formula (13h). In another embodiment, the PG₂ may        be, inter alia, TIPS.

In one embodiment of the invention, the compound of formula (13g) may beobtained by a process including, inter alia, the step of:

-   -   deprotecting the amine of a compound represented by the        structure of formula (13k):

-   -   wherein PG and PG₁ are hydroxy protecting groups, and    -   PG₃ is an amino protecting group,    -   and reacting with an acid form of R₆, thereby obtaining the        compound of formula (13g). In another embodiment, the amino        protecting group may be, inter alia, tBoc.

In one embodiment of the invention, an “alkyl” group refers to asaturated aliphatic hydrocarbon, including straight-chain,branched-chain and cyclic alkyl groups. In one embodiment, the alkylgroup has 1-30 carbons. In another embodiment, the alkyl group has 1-25carbons. In another embodiment, the alkyl group has 1-20 carbons. Inanother embodiment, the alkyl group has 1-10 carbons. In anotherembodiment, the alkyl group has 1-5 carbons. In another embodiment, thealkyl group has 10-25 carbons. In another embodiment, the alkyl grouphas 15-25 carbons. The alkyl group may be unsubstituted or substitutedby one or more groups selected from halogen, hydroxy, alkoxy carbonyl,amido, alkylamido, dialkylamido, nitro, amino, alkylamino, dialkylamino,carboxyl, thio and thioalkyl.

The term alkyl as used throughout the specification and claims includesboth “unsubstituted alkyls” and/or “substituted alkyls”, the latter ofwhich may refer to alkyl moieties having substituents replacing ahydrogen on one or more carbons of the hydrocarbon backbone. Suchsubstituents may include, for example, a halogen, a hydroxyl, analkoxyl, a silyloxy, a carbonyl, and ester, a phosphoryl, an amine, anamide, an imine, a thiol, a thioether, a thioester, a sulfonyl, anamino, a nitro, or an organometallic moiety. It will be understood bythose skilled in the art that the moieties substituted on thehydrocarbon chain may themselves be substituted, if appropriate. Forinstance, the substituents of a substituted alkyl may includesubstituted and unsubstituted forms of amines, imines, amides,phosphoryls (including phosphonates and phosphines), sulfonyls(including sulfates and sulfonates), and silyl groups, as well asethers, thioethers, selenoethers, carbonyls (including ketones,aldehydes, carboxylates, and esters), —CF₃, and —CN. Of course othersubstituents may be applied. In another embodiment, cycloalkyls may befurther substituted with alkyls, alkenyls, alkoxys, thioalkyls,aminoalkyls, carbonyl-substituted alkyls, CF₃, and CN. Of course othersubstituents may be applied. In certain embodiments, the alkyl will beotherwise unsubstituted but will terminate in aryl, substituted aryl,heteroaryl or substituted heteroaryl. The term “unsaturated alkyl” willappear herein interchangeably with the term “alkenyl”; both refer to thesame thing. Thus the residue —CH═CH(CH₂)₁₀CH₃ can be called either“unsaturated alkyl” or “alkenyl”.

Alkoxy or alkoxyl refers to groups of from 1 to 8 carbon atoms of astraight, branched, cyclic configuration and combinations thereofattached to the parent structure through an oxygen. Examples includemethoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, cyclohexyloxy andthe like. Lower-alkoxy refers to groups containing one to four carbons.Lower-alkoxy refers to groups containing one to four carbons. Methoxy ispreferred. For the purpose of this application, alkoxy and lower alkoxyinclude methylenedioxy and ethylenedioxy.

Oxaalkyl refers to alkyl residues in which one or more carbons (andtheir associated hydrogens) have been replaced by oxygen. Examplesinclude methoxypropoxy, 3,6,9-trioxadecyl and the like. The termoxaalkyl is intended as it is understood in the art [see Naming andIndexing of Chemical Substances for Chemical Abstracts, published by theAmerican Chemical Society, ¶196, but without the restriction of¶127(a)], i.e. it refers to compounds in which the oxygen is bonded viaa single bond to its adjacent atoms (forming ether bonds); it does notrefer to doubly bonded oxygen, as would be found in carbonyl groups.

Acyl refers to groups of from 1 to 8 carbon atoms of a straight,branched, cyclic configuration, saturated, unsaturated and aromatic andcombinations thereof, attached to the parent structure through ancarbonyl functionality. One or more carbons in the acyl residue may bereplaced by nitrogen, oxygen or sulfur as long as the point ofattachment to the parent remains at the carbonyl. Examples includeacetyl, benzoyl, propionyl, isobutyryl, t-butoxycarbonyl,benzyloxycarbonyl and the like. Lower-acyl refers to groups containingone to four carbons.

Aryl and heteroaryl mean a 5- or 6-membered aromatic or heteroaromaticring containing 0-3 heteroatoms selected from O, N, or S; a bicyclic 9-or 10-membered aromatic or heteroaromatic ring system containing 0-3heteroatoms selected from O, N, or S; or a tricyclic 13- or 14-memberedaromatic or heteroaromatic ring system containing 0-3 heteroatomsselected from O, N, or S. The aromatic 6- to 14-membered carbocyclicrings include, e.g., benzene, naphthalene, indane, tetralin, andfluorene and the 5- to 10-membered aromatic heterocyclic rings include,e.g., imidazole, pyridine, indole, thiophene, benzopyranone, thiazole,furan, benzimidazole, quinoline, isoquinoline, quinoxaline, pyrimidine,pyrazine, tetrazole and pyrazole.

The term “halogen” means fluorine, chlorine, bromine or iodine.

Any numerical values recited herein include all values from the lowervalue to the upper value in increments of one unit provided that thereis a separation of at least 2 units between any lower value and anyhigher value. As an example, if it is stated that the amount of acomponent or a value of a process variable such as, for example,temperature, pressure, time and the like is, for example, from 1 to 90,preferably from 20 to 80, more preferably from 30 to 70, it is intendedthat values such as 15 to 85, 22 to 68, 43 to 51, 30 to 32 etc. areexpressly enumerated in this specification. For values which are lessthan one, one unit is considered to be 0.0001, 0.001, 0.01 or 0.1 asappropriate. These are only examples of what is specifically intendedand all possible combinations of numerical values between the lowestvalue and the highest value enumerated are to be considered to beexpressly stated in this application in a similar manner.

The abbreviations Me, Et, Ph, Tf, Ts and Ms represent methyl, ethyl,phenyl, trifluoromethanesulfonyl, toluensulfonyl and methanesulfonylrespectively. A comprehensive list of abbreviations utilized by organicchemists (i.e. persons of ordinary skill in the art) appears in thefirst issue of each volume of the Journal of Organic Chemistry. Thelist, which is typically presented in a table entitled “Standard List ofAbbreviations” is incorporated herein by reference.

As used herein, and as would be understood by the person of skill in theart, the recitation of “a compound” is intended to include salts,solvates and inclusion complexes of that compound. For example, thesulfonate (SO₃ ⁻) residue will often appear in formulae herein. As willbe understood by the person of skill in the art, there is an implicationof a counterion, which can be H⁺ (i.e. the acid form) or any cationdiscussed below. Cations of alkali metals and alkaline earth metals, aswell as ammonium cations, are common. Similarly the carboxylate may bewritten COOH, but the salt form COO⁻M⁺ is included within the scope ofthe invention.

Unless chirality is specifically indicated, claims to a compound includeany stereoisomeric form, or a mixture of any such forms of that compoundin any ratio. The term “solvate” refers to a compound in the solidstate, wherein molecules of a suitable solvent are incorporated in thecrystal lattice. A suitable solvent for therapeutic administration isphysiologically tolerable at the dosage administered. Examples ofsuitable solvents for therapeutic administration are ethanol and water.When water is the solvent, the solvate is referred to as a hydrate. Ingeneral, solvates are formed by dissolving the compound in theappropriate solvent and isolating the solvate by cooling or using anantisolvent. The solvate is typically dried or azeotroped under ambientconditions. Inclusion complexes are described in Remington: The Scienceand Practice of Pharmacy 19th Ed. (1995) volume 1, page 176-177, whichis incorporated herein by reference. The most commonly employedinclusion complexes are those with cyclodextrins, and all cyclodextrincomplexes, natural and synthetic, are specifically encompassed withinthe claims.

The term “pharmaceutically acceptable salt” refers to salts preparedfrom pharmaceutically acceptable non-toxic acids or bases includinginorganic acids and bases and organic acids and bases. When thecompounds contain an acidic function (e.g. a carboxylic acid or sulfonicacid residue, suitable pharmaceutically acceptable base addition saltsfor the compounds of the present invention include metallic salts madefrom aluminum, calcium, lithium, magnesium, potassium, sodium and zinc,or organic salts made from lysine, N,N′-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine(N-methylglucamine) and procaine.

While it may be possible for the compounds of formula (I) to beadministered as the raw chemical, it is preferable to present them as apharmaceutical composition. According to a further aspect, the presentinvention provides a pharmaceutical composition comprising a compound offormula (I) or a pharmaceutically acceptable salt or solvate thereof,together with one or more pharmaceutically carriers thereof andoptionally one or more other therapeutic ingredients. The carrier(s)must be “acceptable” in the sense of being compatible with the otheringredients of the formulation and not deleterious to the recipientthereof.

The formulations include those suitable for oral, parenteral (includingsubcutaneous, intradermal, intramuscular, intravenous andintraarticular), rectal and topical (including dermal, buccal,sublingual and intraocular) administration. The most suitable route maydepend upon the condition and disorder of the recipient. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any of the methods well known in the art of pharmacy. Allmethods include the step of bringing into association a compound offormula (I) or a pharmaceutically acceptable salt or solvate thereof(“active ingredient”) with the carrier which constitutes one or moreaccessory ingredients.

Formulations for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient. Formulations for parenteraladministration also include aqueous and non-aqueous sterile suspensions,which may include suspending agents and thickening agents. Theformulations may be presented in unit-dose of multi-dose containers, forexample sealed ampoules and vials, and may be stored in a freeze-dried(lyophilized) condition requiring only the addition of a sterile liquidcarrier, for example saline, phosphate-buffered saline (PBS) or thelike, immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

According to embodiments of the invention, the phrase “protecting group”as used herein means temporary modifications of a potentially reactivefunctional group which protect it from undesired chemicaltransformations. Examples of such protecting groups include esters ofcarboxylic acids, silyl ethers of alcohols, and acetals and ketals ofaldehydes and ketones, respectively. Of course other appropriateprotecting groups may be used.

In one embodiment of the invention, the protecting group may be, interalia, a hydroxy protecting group. In one embodiment of the invention,the hydroxy protecting group may be, inter alia, an alkyl, aryl,aralkyl, silyl or acyl radical. In another embodiment, the protectinggroup may be, inter alia, trimethylsilyl, triethylsilyl,tert-butyldimethylsilyl (TBS), triisopropylsilyl (TIPS), ortert-butyldiphenylsilyl. Of course, any other appropriate protectinggroup may be used. In one embodiment, the aralkyl may be unsubstitutedor substituted. In another embodiment, the aralkyl may be, inter alia,arylmethyl. In another embodiment, the protecting group may be, interalia, benzyl. In another embodiment, the protecting group may be, interalia, methoxybenzyl. In another embodiment, the methoxybenzyl may be,inter alia, para-methoxybenzyl.

In one embodiment of the invention, the amino protective group may beany of amino protective group (see for example “Protection for the aminogroup” in T. W. Green & P. G. M. Wuts, Protective groups in organicsynthesis, 3rd Ed., 1999, 494-653).

In one embodiment of the invention, the protecting group may be, interalia, an amino protecting group. In one embodiment of the invention, theamino protecting group may be, inter alia, carbamate, an amide or anN-sulfonylamide. In another embodiment, the amino protecting group maybe, inter alia, benzyloxycarbonyl (Cbz), 9-fluorenylmethyloxycarbonyl(Fmoc), t-butyloxycarbonyl, (tBoc), biphenylisopropyloxycarbonyl,t-amyloxycarbonyl, isobornyloxycarbonyl,alpha-dimethyl-3,5-dimethoxybenzyloxycarbonyl or2-cyano-t-butyloxycarbonyl. In another embodiment, the amino protectinggroup (PG) may be, inter alia, benzyloxycarbonyl (Cbz).

Furthermore, in one embodiment, the invention provides a pharmaceuticalcomposition including, inter alia, any one of the compounds of thisinvention or any combination thereof, together with one or morepharmaceutically acceptable excipients.

Furthermore, in one embodiment, the invention provides a method forstimulating, inhibiting, suppressing or modulating an immune response ina subject, the method may include, inter alia, administering to asubject any one of the compounds of this invention or any combinationthereof.

Furthermore, in one embodiment, the invention provides a method forstimulating, inhibiting, suppressing or modulating an immune response ina subject, the method includes, inter alia, administering to a subject apharmaceutical composition including, inter alia, any one of thecompounds of this invention or any combination thereof, together withone or more pharmaceutically acceptable excipients.

Furthermore, in one embodiment, “pharmaceutical composition” can mean atherapeutically effective amount of one or more compounds of the presentinvention together with suitable excipients and/or carriers useful forstimulating, inhibiting, suppressing or modulating an immune response ina subject. In one embodiment, “therapeutically effective amount” mayrefer to that amount that provides a therapeutic effect for a givencondition and administration regimen. In one embodiment, suchcompositions can be administered by any method known in the art.

In one embodiment, the compositions of the present invention areformulated as oral or parenteral dosage forms, such as uncoated tablets,coated tablets, pills, capsules, powders, granulates, dispersions orsuspensions. In another embodiment, the compositions of the presentinvention are formulated for intravenous administration. In anotherembodiment, the compounds of the present invention are formulated inointment, cream or gel form for transdermal administration. In anotherembodiment, the compounds of the present invention are formulated as anaerosol or spray for nasal application. In another embodiment, thecompositions of the present invention are formulated in a liquid dosageform. Examples of suitable liquid dosage forms include solutions orsuspensions in water, pharmaceutically acceptable fats and oils,alcohols or other organic solvents, including esters, emulsions, syrupsor elixirs, solutions and/or suspensions.

Suitable excipients and carriers may be, according to embodiments of theinvention, solid or liquid and the type is generally chosen based on thetype of administration being used. Liposomes may also be used to deliverthe composition. Examples of suitable solid carriers include lactose,sucrose, gelatin and agar. Oral dosage forms may contain suitablebinders, lubricants, diluents, disintegrating agents, coloring agents,flavoring agents, flow-inducing agents, and melting agents. Liquiddosage forms may contain, for example, suitable solvents, preservatives,emulsifying agents, suspending agents, diluents, sweeteners, thickeners,and melting agents Parenteral and intravenous forms should also includeminerals and other materials to make them compatible with the type ofinjection or delivery system chosen. Of course, other excipients mayalso be used.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates structures of α-galactosylceramide, sulfatide and3-O-sulfo-α/β-galactosylceramides 10, 24, according to embodiments ofthe invention.

FIG. 2 demonstrates the preparation of (a) compound IV, according toembodiments of the invention; (b) the preparation of compound 4,according to embodiments of the invention.

FIG. 3 demonstrates the preparation of compound XV, according toembodiments of the invention.

FIG. 4 demonstrates (a) the preparation of compound XVIII, according toembodiments of the invention; (b) the preparation of compound 10,according to embodiments of the invention.

FIG. 5 depicts structures of glycolipids and analogues thereof,according to embodiments of the invention.

FIG. 6 schematically depicts the synthesis of sphingosine, according toembodiments of the invention, as conducted herein. Reagents andconditions; a) C2H3MgBr, THF, Anti:Syn 3.5:1 61%; b) (i) Grubbs catalyst2nd generation, CH2Cl2, Pentadecene, 71%; (ii) BzCl, pyridine, 90%;(iii) Amberlyst 15 H+ form, MeOH 70%.

FIG. 7 schematically depicts the synthesis of some glycolipids,according to embodiments of the invention, as conducted herein. Reagentsand conditions; a) 32, TMSOTf, 67%; b) (i) TFA, DCM, (ii) HBTU, myristicacid or 2-(S)-hydroxy myristic acid, n-Morpholine, ≈92% 2 steps; c) H₂,20% Pd(OH)2, (ii) LiOH, H2O:THF:MeOH, 38%, 2 steps; d) 36, TMSOTf, 60%;e) (i) TFA, DCM, (ii) HBTU, myristic acid or 2-(S)-hydroxy myristicacid, n-Morpholine, ≈92% 2 steps; f) (i) NaOMe, MeOH, (ii) Pd/C, H₂,EtOH, 90% 2 steps; g) 40, TMSOTf, 62%. h) (i) TFA, DCM, (ii) HBTU,nervonic acid, n-Morpholine, 60% 2 steps; i) (i) NaOMe, MeOH, quant.(ii) Bu₂SnO, MeOH, (iii) Me₃N.SO₃, THF, 95%; j-k) (i) LDA, TMSOOTMS,(ii) H+, MeOH, 30%, 2 steps; then LiOH, H₂O:MeOH:THF, 81%; l) Novozyme435, CH₂═CHOAc, 54% based on S isomer.

FIG. 8 demonstrates the IL-2 secretion profiles obtained withglycolipids, according to embodiments of the invention. (a) IL-2secretion profile obtained with 3-O-sulfo-GalCer, as compared toα-GalCer and analogues. (b) Dose dependent secretion of IL-2 bySphingomonas GSLs and analogues.

FIG. 9 demonstrates human NKT cell responses to glycolipids, accordingto embodiments of the invention. Human Vα24i NKT cells respond tosynthetic Sphingomonas and sulfatide glycolipids, in terms of IFN-γ (a)and IL-4 (b) release after culture with 4×10⁵ autologous immature CD14+dendritic cells pulsed with the indicated glycolipid antigens at 10μg/ml. Negative controls included similar numbers of NKT cells anddendritic cells, cultured without added glycolipid. Data representmean±S.D. of duplicate well; (c) in vitro INF-γ secretion by humanCD161⁺ NK+NKT cells (2×10⁵/well) in the presence of CD14⁺ DCs(4×10⁵/well) and 20 μg/ml of various glycolipids; (d) in vitro IL-4secretion by human CD161⁺ NK+NKT cells (2×10⁵/well) in the presence ofCD14⁺ DCs (4×10⁵/well) and 20 μg/ml of various glycolipids.

FIG. 10 demonstrates a flow cytometric analysis of a human Vα24i humanNKT cell line with human CD1d dimers that were unloaded or loaded with10M of the indicated glycolipid antigen, according to embodiments of theinvention. The cells were also stained with anti-human CD3-PerCP.

FIG. 11 depicts a computer-generated model of GSL-1 docked to thecrystal structure of mCD1d, according to embodiments of the invention.The two acyl tails fit nicely into the hydrophobic pockets of theprotein allowing for the sugar head group to be presented for NKT cellrecognition.

FIG. 12 demonstrates IL-2 secretion profiles obtained from murine NKTcells presented with the glycolipids as indicated, according toembodiments of the invention.

FIG. 13 demonstrates IL-2 secretion profiles obtained from murine NKTcells presented with other glycolipids as indicated, according toembodiments of the invention.

FIGS. 14, 15 and 16 demonstrate IFN-γ secretion from human NKT cellspresented with the glycolipids as indicated, supplied at the indicatedconcentration.

FIGS. 17 and 18 demonstrate IFN-γ secretion from human NKT cellspresented with the glycolipids as indicated, supplied at higherconcentration.

FIGS. 19A-C demonstrate similar IFN-γ secretion from human NKT cellspresented with the glycolipids, at the indicated concentration, in thecontext of Hela cells transfected with CD1d.

FIG. 20 demonstrates IL-4 secretion from human NKT cells presented withthe glycolipids, at the indicated concentration in the context ofdendritic cells (A) or transfected Hela cells (B).

FIG. 21 demonstrates multi-cytokine secretion from human NKT cellspresented with the glycolipids, at the indicated concentration in thecontext of dendritic cells: (A)/(B) 1010A and 1010B NKT cells—IFN-γ;(C)/(D) 1010A and 1010B NKT cells—IL-12; (E)/(F) 1010A and 1010B NKTcells—GMCSF.

FIG. 22 demonstrates in vivo secretion from B6 mice presented withindicated glycolipids: (A) IFN-γ; (B) IL-4; (C) IL-12.

FIG. 23(A) depicts the superimposition of docking results of compound 84from Example 10 (yellow) with the crystal structure of α-GalCer(green)/hCD1d complex. The α2 helix is removed for clarity. The overallbinding motif of the docked compound did not notably deviate from thecrystallized structure. The terminal phenyl group 84 is within distanceto interact with the aromatic ring of Tyr73; (B) depicts the hCD1ddocking model of analogue compounds 83-85.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

This invention provides, in one embodiment, a compound represented bythe structure of formula 1:

-   -   wherein, R═COOR₁ or CH₂OR₁;        -   R₁═H or an alkyl group;        -   R₂═H or SO₃ ⁻;        -   R₃═H or OH;        -   R₃′═H or OH;        -   R₄ and R₄′ are independently chosen from        -   (a) H,        -   (b) alkyl;        -   (c) alkenyl; and        -   (d) oxaalkyl;        -   R₅═OH, acetamido or a halogen atom;        -   or a pharmaceutically acceptable salt thereof,        -   wherein if R═CH₂OR₁, R₂═H, R₃ is OH and R₃′ is H, then one            of the following is true:        -   (i) R₅=acetamido, halogen atom or OH in an axial position;        -   (ii) R₄═H, or unsaturated or saturated alkyl chain having 9            carbon atoms or fewer; or        -   (iii) R₄′═H, or unsaturated or saturated alkyl chain having            20 carbon atoms or fewer.

In another embodiment, this invention provides a compound represented bythe structure of formula 2:

-   -   wherein R═COOR₁ or CH₂OR₁;        -   R₁═H or an alkyl group;        -   R₂═H or SO₃ ⁻;        -   R₃═H or OH;        -   R₃′═H or OH; and        -   R₄═H, unsaturated or saturated, alkyl group; and        -   R₄′═H, unsaturated or saturated, alkyl group;    -   or a pharmaceutically acceptable salt thereof,        wherein if R═CH₂OR₁, R₂═H, R₃ is OH and R₃′ is H, then one of        the following is true:    -   (i) R₄═H, or unsaturated or saturated alkyl chain having 9        carbon atoms or fewer;        or (ii) R₄′═H, or unsaturated or saturated alkyl chain having 20        carbon atoms or fewer.

In one embodiment, the alkyl chain of R₄ has 1 carbon atom, in anotherembodiment, the alkyl chain of R₄ has between 1-5, or in anotherembodiment, 2-6, or in another embodiment, 3-7, or in anotherembodiment, 4-8, or in another embodiment 5-9 carbon atoms. In oneembodiment, the alkyl chain of R₄ has 10-25 carbon atom, in anotherembodiment, the alkyl chain of R₄ has between 10-15 carbon atoms.

In another embodiment, the alkyl chain of R₄′ has 1 carbon atom, inanother embodiment, the alkyl chain of R₄′ has between 1-10, or inanother embodiment, 10-15, or in another embodiment, 5-13, or in anotherembodiment, 8-15, or in another embodiment 10-25 carbon atoms or, inanother embodiment, between 20-30 carbon atoms.

In another embodiment, this invention provides a compound represented bythe structure of formula 3:

-   -   wherein, R═COOR₁ or CH₂OR₁;        -   R₁═H or an alkyl group;        -   R₂═SO₃ ⁻; and        -   n=integer;    -   or a pharmaceutically acceptable salt thereof.

In another embodiment, n is an integer ranging from 1-5, or, in anotherembodiment, between 5-10, or in another embodiment, 10-15, or in anotherembodiment, 10-20, or in another embodiment, 1-15, or in anotherembodiment 15-25 carbon atoms or, in another embodiment, between 10-30.

In another embodiment, this invention provides a compound represented bythe structure of formula 4:

-   -   or a pharmaceutically acceptable salt thereof.

In another embodiment, the salt may be, inter alia, a sodium salt.

In another embodiment, this invention provides a compound represented bythe structure of formula 5:

In another embodiment, this invention provides a compound represented bythe structure of formula 6:

In another embodiment, this invention provides a compound represented bythe structure of formula 7:

In another embodiment, this invention provides a compound represented bythe structure of formula 8:

In one embodiment, this invention provides a compound represented by thestructure of formula 9:

-   -   wherein, R═COOR₁ or CH₂OR₁        -   R₁═H or an alkyl group;        -   R₂═H or SO₃ ⁻;        -   R₃═OH;        -   R₃′═H or OH; and        -   R₄═H, unsaturated or saturated, alkyl group; and        -   R₄′═H, unsaturated or saturated, alkyl group;            -   or a pharmaceutically acceptable salt thereof,                wherein if R═CH₂OR₁, R₂═H, R₃ is OH and R₃′ is H, then                one of the following is true:    -   (i) R₄═H, or unsaturated or saturated alkyl chain having 9        carbon atoms or fewer; or    -   (ii) R₄′═H, or unsaturated or saturated alkyl chain having 20        carbon atoms or fewer.

In another embodiment, this invention provides a compound represented bythe structure of formula 10:

or a pharmaceutically acceptable salt thereof. In another embodiment,the salt may be, inter alia, a sodium salt.

In still another embodiment, this invention provides a compoundrepresented by the structure of formula 17:

-   -   wherein    -   R₄ and R₆ are independently selected from:        -   (a) alkyl,        -   (b) alkenyl,        -   (c) alkyl terminating in aryl, substituted aryl, heteroaryl            or substituted heteroaryl; and        -   (d) alkenyl terminating in aryl, substituted aryl,            heteroaryl or substituted heteroaryl.

In certain embodiments of compounds of formula 17,

-   -   R₆ is

-   -   X is an alkyl chain; and    -   R₇ is chosen from halogen, H, phenyl, alkyl, alkoxy, nitro, and        CF₃.

In other embodiments of compounds of formula 17,

-   -   R₄ is

-   -   X is an alkyl chain; and

R₇ is chosen from halogen, H, phenyl, alkyl, alkoxy, nitro, and CF₃.

Furthermore, in one embodiment, the invention provides a process for thepreparation of a compound represented by the structure of formula (4)

-   -   or a pharmaceutically salt thereof, the process including, inter        alia, the step of:        -   removing the benzyldiene protecting group and hydrogenating            of the compound represented by the structure of formula            (4a),

or a salt thereof, wherein PG is a hydroxy protecting group. In anotherembodiment, the hydroxy protecting group may be benzyl.

In one embodiment of the invention, the compound of formula (4a) may beobtained by a process including, inter alia, the step of:

-   -   conducting a selective sulfation of the 3″ OH of the galactose        moiety of the compound represented by the structure of formula        (4b):

wherein PG is a hydroxy protecting group and R is H. In anotherembodiment, the hydroxy protecting group may be benzyl.

In one embodiment of the invention, the compound of formula (4b) whereinR is H, may be obtained by a process including, inter alia, the step ofremoving the levulinyl protecting group of a compound of formula (4b)wherein R is levulinyl, thereby obtaining a compound of formula (4b)wherein R is H.

In one embodiment of the invention, the compound of formula (4b) whereinR is levulinyl may be obtained by a process including, inter alia, thestep of:

-   -   reacting a compound represented by the structure of formula        (4c):

wherein R is H or levulinyl with hexacosanoic acid, thereby obtainingthe compound of formula (4b) wherein R is levulinyl.

In one embodiment of the invention, the compound of formula (4c),wherein R is H or levulinyl, may be obtained by a process including,inter alia, the step of:

-   -   reducing the azide group of a compound represented by the        structure of formula (4d):

-   -   wherein R is levulinyl, thereby obtaining a compound of formula        (4c) wherein R is H or levulinyl.

In one embodiment of the invention, the compound of formula (4d) whereinR is levulinyl, may be obtained by a process including, inter alia, thestep of:

-   -   reacting a compound represented by the structure of formula (4e)

-   -   wherein PG is a hydroxy protecting group, LG is a leaving group        and R is levulinyl,    -   with a compound represented by the structure of formula (4f)

-   -   wherein PG is a hydroxy protecting group,    -   to form an alpha glycosidic bond, thereby obtaining the compound        of formula (4d) wherein R is levulinyl. In another embodiment,        the leaving group may be, inter alia,

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of formula (10)

-   -   or a pharmaceutically salt thereof, including, inter alia, the        step of:    -   conducting a selective sulfation of the 3″ OH of the galactose        moiety of the compound represented by the structure of formula        (10a):

-   -   In another embodiment, the sulfation may be conducted in the        presence of Bu₂SnO.

In one embodiment of the invention, the compound of formula (10a) may beobtained by the process including, inter alia, the step of:

-   -   removing the hydroxy protecting groups and hydrogenating the        compound represented by the structure of formula (10b):

wherein PG and PG₁ are hydroxy protecting groups, thereby obtaining thecompound of formula (10a). In another embodiment, the PG may be, interalia, benzyl. In another embodiment, the PG1 may be, inter alia,benzoyl. In one embodiment of the invention, the compound of formula(10b) may be obtained by a process including, inter alia, the step of:

-   -   reacting a compound represented by the structure of formula        (10c):

-   -   wherein PG is a hydroxy protecting group,    -   with a compound represented by the structure of formula (10d):

wherein PG₁ is a hydroxy protecting group and LG is a leaving group,thereby obtaining the compound of formula (10b). In another embodiment,the leaving group may be, inter alia,

In one embodiment of the invention, the compound of formula (10c) may beobtained by a process comprising the steps of:

-   -   reducing the azide of a compound represented by the structure of        formula (10e):

-   -   wherein PG and PG₂ are hydroxy protecting groups;    -   reacting the resulting amine with hexacosanoic acid; and        removing the hydroxy protecting group PG₂, thereby obtaining the        compound of formula (10c). In another embodiment, the PG₂ may        be, inter alia, TIPS.

In one embodiment, the invention provides a process for the preparationof a compound represented by the structure of formula (11):

-   -   or a pharmaceutically salt thereof, including, inter alia, the        step of:    -   conducting a selective sulfation of the 3″ OH of the galactose        moiety of the compound represented by the structure of formula        (11a):

thereby obtaining the a compound represented by the structure of formula(10). In another embodiment, the sulfation may be conducted in thepresence of Bu₂SnO.

In one embodiment of the invention, the compound of formula (11a) may beobtained by the process including, inter alia, the step of:

-   -   removing the hydroxy protecting groups of the compound        represented by the structure of formula (11b):

wherein PG and PG₁ are hydroxy protecting groups, thereby obtaining thecompound of formula (11a). In another embodiment, PG may be, inter alia,benzoyl. In another embodiment, PG₁ may be, inter alia, benzoyl.

In one embodiment of the invention, the compound of formula (10b) may beobtained by a process including, inter alia, the step of:

-   -   deprotecting the amine of a compound represented by the        structure of formula (11c):

-   -   wherein PG and PG₁ are hydroxy protecting groups, and    -   PG₃ is an amino protecting group,    -   and reacting with nervonic acid, thereby obtaining the compound        of formula (11b). In another embodiment, the amino protecting        group may be, inter alia, tBoc.

In one embodiment, any one of the compounds of the invention may be aligand for an NKT (natural killer T) cell. In another embodiment, theligand may be in a complex with a CD1 molecule. In another embodiment,the CD1 molecule is a CD1d molecule. In another embodiment, the ligandstimulates NKT cells, which express a CD161+ NK marker as well as aninvariant T cell antigen receptor (TCR) on the surface thereof.

In another embodiment, the invention provides a composition or vaccineincluding, inter alia, any one of the compounds of the invention. Inanother embodiment, the composition or vaccine may include, inter alia,at least one cell population. In another embodiment, the cell populationmay include, inter alia, NKT cells, antigen presenting cells, or acombination thereof.

In another embodiment, the invention provides a method for stimulatingNKT cells, the method including, inter alia, contacting an NKT cell withany one of the compounds of the invention.

In another embodiment, the invention provides a cell population obtainedby any one of the methods of the invention.

In another embodiment, the invention provides a method for stimulating,inhibiting, suppressing or modulating an immune response in a subject,the method includes, inter alia, the step of contacting an NKT cell inthe subject with any one of the compounds of the invention.

In another embodiment, the compound according to the invention may be ina complex with a CD1 molecule. In another embodiment, the CD1 moleculemay be CD1d. In another embodiment, the complex may be displayed on adendritic cell. In another embodiment, the complex may be displayed onany antigen presenting cell.

In one embodiment of the invention, the NKT cells secrete a cytokine. Inanother embodiment the NKT cell may be a Vα24iNKT cell in humans. Inanother embodiment the NKT cell may be a Vα14i NKT cell in mice.

In one embodiment of the invention, the subject may beimmunocompromised. In another embodiment, the subject is infected. Inanother embodiment, the subject is infected with HIV. In anotherembodiment, the subject is infected with mycobacteria. In anotherembodiment, the subject is infected with malaria. In another embodiment,the subject is infected with HIV, mycobacteria, or malaria.

In one embodiment of the invention, the subject is afflicted withcancer. In one embodiment of the invention, the subject is at anelevated risk for cancer. In one embodiment of the invention, thesubject has precancerous precursors.

In one embodiment of the invention, the immune response is biased towardTh1 or Th2. In another embodiment, the subject suffers from, or is at anelevated risk for an autoimmune disease. In another embodiment, thebiasing of the immune response results in the suppression, inhibition orabrogation of the autoimmune disease. In another embodiment, the subjecthas an inappropriate or undesirable immune response. In anotherembodiment, the response is inflammatory. In another embodiment, theinappropriate or undesirable response exacerbates an infection, diseaseor symptom in the subject.

In another embodiment, the invention provides an adjuvant including,inter alia, any one of the compounds according to the invention.

In another embodiment, the invention provides a method of enhancingimmunogenicity of a compound, composition, or vaccine in a subject, themethod includes, inter alia, administering to the subject a compound,composition or vaccine further comprising an adjuvant of according tothe invention, wherein the adjuvant enhances the immunogenicity of thecompound, composition or vaccine.

In another embodiment, the invention provides a method of stimulating orenhancing cytokine production in a subject, the method includes, interalia, administering to the subject any one of the compounds of theinvention, whereby an NKT cell in the subject secretes a cytokinefollowing contact with the compound. In another embodiment, the cytokinemay be interferon-γ or Interleukin-4.

In another embodiment, this invention provides an NK T cell obtained viacontacting an NK T cell with a compound of this invention. In oneembodiment, such contact is in the presence of an antigen presentingcell, which in another embodiment expresses a CD1 molecule, wherein thecompound, or a fragment thereof, is displayed in the context of the CD 1molecule.

In one embodiment, the phrase “NKT cell” or “Natural Killer cell”,refers to a T cell population that causes, stimulates or contributes tocytokine production, and/or in another embodiment, is cytotoxic. In oneembodiment, the NKT cells are a homogenous population, or in anotherembodiment, a heterogeneous population.

NKT cells are an exceptional subset of mature lymphocytes that bear bothNK and T cell receptors. Murine NKT cells express NK1.1 and TCRαβreceptors and are especially dense in the bone marrow and liver. Thecells may express a very limited TCR repertoire, which may include aninvariant α-chain. The ligand for NKT cells may be non-polymorphic, anda non-classical MHC class I molecule may present a specific antigenprocessed via a TAP (transporter associated with antigenprocessing)-independent pathway.

In one embodiment, the antigen is presented in the context of a CD1molecule, which in one embodiment is CD1d. Activated NK T cells maydisplay an NK-like perforin-dependent cytotoxicity against variouscells, including tumor cells or cell lines and inhibit tumor metastasis,among other applications, as is described further hereinbelow, andrepresenting embodiments of the methods of this invention.

The T cells of this invention may express CD161 and Vα24i TCR on theircell surface. In one embodiment, the T cells may be classified as CD161^(high) expressors, or in another embodiment, the T cells may beclassified as CD 161^(low) expressors, or in another embodiment, acombination thereof.

It is to be understood that the NK T cells of this invention, and thoseobtained via the methods of this invention, may express any number orcombination of cell surface markers, as described herein, and as is wellknown in the art, and are to be considered as part of this invention.

In one embodiment, the T cell subpopulation, are “invariant NK T cells,”which may represent a major fraction of the mature T cells in thymus,the major T cell subpopulation in murine liver, and/or up to 5% ofsplenic T cells.

In another embodiment, the T cell subpopulation may be “non-invariant NKT cells”, which may comprise human and mouse bone marrow and human liverT cell populations that are, for example, CD1d-reactive noninvariant Tcells which express diverse TCRs, and which can also produce a largeamount of IL-4 and IFN-γ.

In one embodiment, the NKT cells of this invention are obtained bypositive selection for expression of CD161 and Vα24i TCR, and in anotherembodiment, the T cells may be obtained via negative selectionprocedures, as are well known in the art.

In one embodiment, the NK T cells of this invention may be obtained fromin vivo sources, such as, for example, peripheral blood, leukopheresisblood product, apheresis blood product, peripheral lymph nodes, gutassociated lymphoid tissue, spleen, thymus, cord blood, mesenteric lymphnodes, liver, sites of immunologic lesions, e.g. synovial fluid,pancreas, cerebrospinal fluid, tumor samples, granulomatous tissue, orany other source where such cells may be obtained. In one embodiment,the NK T cells are obtained from human sources, which may be, in anotherembodiment, from human fetal, neonatal, child, or adult sources. Inanother embodiment, the NK T cells of this invention may be obtainedfrom animal sources, such as, for example, porcine or simian, or anyother animal of interest. In another embodiment, the NK T cells of thisinvention may be obtained from subjects that are normal, or in anotherembodiment, diseased, or in another embodiment, susceptible to a diseaseof interest.

In one embodiment, the T cells and/or cells, as described furtherhereinbelow, of this invention are isolated from tissue, and, in anotherembodiment, an appropriate solution may be used for dispersion orsuspension, toward this end. In another embodiment, T cells and/orcells, as described further hereinbelow, of this invention may becultured in solution.

Such a solution may be, in another embodiment, a balanced salt solution,such as normal saline, PBS, or Hank's balanced salt solution, or others,each of which represents another embodiment of this invention. Thesolution may be supplemented, in other embodiment, with fetal calfserum, bovine serum albumin (BSA), normal goat serum, or other naturallyoccurring factors, and, in another embodiment, may be supplied inconjunction with an acceptable buffer. The buffer may be, in otherembodiments, HEPES, phosphate buffers, lactate buffers, or the like, aswill be known to one skilled in the art.

In another embodiment, the solution in which the T cells or cells ofthis invention may be placed is in medium is which is serum-free, whichmay be, in another embodiment, commercially available, such as, forexample, animal protein-free base media such as X-VIVO 10™ or X-VIVO 15™(BioWhittaker, Walkersville, Md.), Hematopoietic Stem Cell-SFM media(GibcoBRL, Grand Island, N.Y.) or any formulation which promotes orsustains cell viability. Serum-free media used, may, in anotherembodiment, be as those described in the following patent documents: WO95/00632; U.S. Pat. No. 5,405,772; PCT US94/09622. The serum-free basemedium may, in another embodiment, contain clinical grade bovine serumalbumin, which may be, in another embodiment, at a concentration ofabout 0.5-5%, or, in another embodiment, about 1.0% (w/v). Clinicalgrade albumin derived from human serum, such as Buminate® (BaxterHyland, Glendale, Calif.), may be used, in another embodiment.

In another embodiment, the T cells of this invention may be separatedvia affinity-based separation methods. Techniques for affinityseparation may include, in other embodiments, magnetic separation, usingantibody-coated magnetic beads, affinity chromatography, cytotoxicagents joined to a monoclonal antibody or use in conjunction with amonoclonal antibody, for example, complement and cytotoxins, and“panning” with an antibody attached to a solid matrix, such as a plate,or any other convenient technique. In other embodiment, separationtechniques may also include the use of fluorescence activated cellsorters, which can have varying degrees of sophistication, such asmultiple color channels, low angle and obtuse light scattering detectingchannels, impedance channels, etc. It is to be understood that anytechnique, which enables separation of the NK T cells of this inventionmay be employed, and is to be considered as part of this invention.

In another embodiment, the affinity reagents employed in the separationmethods may be specific receptors or ligands for the cell surfacemolecules indicated hereinabove.

In another embodiment, the antibodies utilized herein may be conjugatedto a label, which may, in another embodiment, be used for separation.Labels may include, in other embodiments, magnetic beads, which allowfor direct separation, biotin, which may be removed with avidin orstreptavidin bound to, for example, a support, fluorochromes, which maybe used with a fluorescence activated cell sorter, or the like, to allowfor ease of separation, and others, as is well known in the art.Fluorochromes may include, in one embodiment, phycobiliproteins, suchas, for example, phycoerythrin, allophycocyanins, fluorescein, Texasred, or combinations thereof.

In one embodiment, cell separations utilizing antibodies will entail theaddition of an antibody to a suspension of cells, for a period of timesufficient to bind the available cell surface antigens. The incubationmay be for a varied period of time, such as in one embodiment, for 5minutes, or in another embodiment, 15 minutes, or in another embodiment,30 minutes, or in another embodiment, 45 minutes, or in anotherembodiment, 60 minutes, or in another embodiment, 90 minutes. Any lengthof time which results in specific labeling with the antibody, withminimal non-specific binding is to be considered envisioned for thisaspect of the invention.

Any length of time which results in specific labeling with the antibody,with minimal non-specific binding is to be considered envisioned forthis aspect of the invention.

In another embodiment, the staining intensity of the cells can bemonitored by flow cytometry, where lasers detect the quantitative levelsof fluorochrome (which is proportional to the amount of cell surfaceantigen bound by the antibodies). Flow cytometry, or FACS, can also beused, in another embodiment, to separate cell populations based on theintensity of antibody staining, as well as other parameters such as cellsize and light scatter.

In another embodiment, the labeled cells are separated based on theirexpression of CD161 and Vα24i TCR. The separated cells may be collectedin any appropriate medium that maintains cell viability, and may, inanother embodiment, comprise a cushion of serum at the bottom of thecollection tube.

In another embodiment, the culture containing the T cells of thisinvention may contain other cytokines or growth factors to which thecells are responsive. In one embodiment, the cytokines or growth factorspromote survival, growth, function, or a combination thereof of the NK Tcells. Cytokines and growth factors may include, in other embodiment,polypeptides and non-polypeptide factors.

In one embodiment, the NK T cell populations of this invention areantigen specific.

In one embodiment, the term “antigen specific” refers to a property ofthe population such that supply of a particular antigen, or in anotherembodiment, a fragment of the antigen, results, in one embodiment, inspecific cell proliferation, when presented the antigen, which in oneembodiment, is in the context of CD1. In one embodiment, the antigen isany compound of this invention.

In another embodiment, supply of the antigen or fragment thereof,results in NK T cell production of interleukin 2, or in anotherembodiment, interferon-γ, or in another embodiment, interleukin-4, or inanother embodiment, a combination thereof. In one embodiment, the NK Tcell population expresses a monoclonal T cell receptor. In anotherembodiment, the NK T cell population expresses polyclonal T cellreceptors.

In one embodiment, the T cells will be of one or more specificities, andmay include, in another embodiment, those that recognize a mixture ofantigens derived from an antigenic source. In one embodiment, a mixtureof the compounds of this invention may be used to simulate a NK T cellsof varying specificity.

In one embodiment, the NK T cell population suppresses an autoimmuneresponse. In one embodiment, the term “autoimmune response” refers to animmune response directed against an auto- or self-antigen. In oneembodiment, the autoimmune response is rheumatoid arthritis, multiplesclerosis, diabetes mellitus, myasthenia gravis, pernicious anemia,Addison's disease, lupus erythematosus, Reiter's syndrome, atopicdermatitis or Graves disease. In one embodiment, the autoimmune diseasecaused in the subject is a result of self-reactive T cells, whichrecognize multiple self-antigens.

In another embodiment, the NK T cell population suppresses aninflammatory response. In one embodiment, the term “inflammatoryresponse” refers to any response that is, in one embodiment, caused byinflammation or, in another embodiment, whose symptoms includeinflammation. By way of example, an inflammatory response may be aresult of septic shock, or, in another embodiment, a function ofrheumatoid arthritis. The inflammatory response may be a part of anoverall inflammatory disorder in a subject, and may comprise, in anotherembodiment, cardiovascular disease, rheumatoid arthritis, multiplesclerosis, Crohn's disease, inflammatory bowel disease, systemic lupuserythematosis, polymyositis, septic shock, graft versus host disease,host versus graft disease, asthma, rhinitis, psoriasis, cachexiaassociated with cancer, or eczema. In one embodiment, as describedhereinabove, the inflammation in the subject may be a result of T cells,which recognize multiple antigens in the subject. In one embodiment, theNK T cells of this invention may be specific for a single antigen wheremultiple antigens are recognized, yet the NK T cell populationeffectively suppresses inflammation in the subject. In one embodiment,suppression of inflammation is via modulating an immune response as aresult of production of a particular cytokine profile. In oneembodiment, the NK T cells produce cytokines which serve to downmodulatethe inflammatory response.

In another embodiment, the NK T cell populations of this inventionsuppresses an allergic response. In one embodiment, the term “allergicresponse” refers to an immune system attack against a generallyharmless, innocuous antigen or allergen. Allergies may in one embodimentinclude, but are not limited to, hay fever, asthma, atopic eczema aswell as allergies to poison oak and ivy, house dust mites, bee pollen,nuts, shellfish, penicillin or other medications, or any other compoundor compounds which induce an allergic response. In one embodiment,multiple allergens elicit an allergic response, and the antigenrecognized by the NK T cells of this invention may be any antigenthereof. In one embodiment, suppression of allergic responses is viamodulating an immune response as a result of production of a particularcytokine profile. In one embodiment, the NK T cells produce cytokineswhich serve to downmodulate the allergic response.

In another embodiment, the NK T cells of the present invention areutilized, in circumstances wherein eliciting a “Th1” response isbeneficial in a subject, wherein the subject has a disease where aso-called “Th2” type response has developed. Introduction of the NK Tcells, in one embodiment, results in a shift toward a Th1 type response,in response to the cytokine profile produced from the NK T cells.

In one embodiment, the term “Th2 type response” refers to a pattern ofcytokine expression, elicited by T Helper cells as part of the adaptiveimmune response, which support the development of a robust antibodyresponse. Typically Th2 type responses are beneficial in helminthinfections in a subject, for example. Typically Th2 type responses arerecognized by the production of interleukin-4 or interleukin 10, forexample.

In one embodiment, the term “Th1 type response” refers to a pattern ofcytokine expression, elicited by T Helper cells as part of the adaptiveimmune response, which support the development of robust cell-mediatedimmunity. Typically Th1 type responses are beneficial in intracellularinfections in a subject, for example. Typically Th1 type responses arerecognized by the production of interleukin-2 or interferon γ, forexample.

In another embodiment, the reverse occurs, where a Th1 type response hasdeveloped, when Th2 type responses provide a more beneficial outcome toa subject, where introduction of the NK T cells, vaccines orcompositions of the present invention provides a shift to the morebeneficial cytokine profile. One example would be in leprosy, where theNK T cells, vaccines or compositions of the present invention stimulatesa Th1 cytokine shift, resulting in tuberculoid leprosy, as opposed tolepromatous leprosy, a much more severe form of the disease, associatedwith Th2 type responses.

In another embodiment, the NK T cells of this invention, and obtainedvia the methods of this invention, may be a part of a vaccine orcomposition. Such vaccines and/or compositions may be used in anyapplicable method of this invention, and represents an embodimentthereof.

For example, in one embodiment, the methods of this invention forstimulating, inhibiting, suppressing or modulating an immune response ina subject, which comprise contacting an NKT cell in a subject with acompound of the invention, may also comprise contacting the NKT cellwith a compound in a composition, or in another embodiment, contactingthe NKT cell with a vaccine comprising at least one compound of theinvention.

It is to be understood that any use of the NK T cells, vaccines orcompositions of the present invention for methods of enhancingimmunogenicity, such as, for example, for purposes of immunizing asubject to prevent disease, and/or ameliorate disease, and/or alterdisease progression are to be considered as part of this invention.

Examples of infectious virus to which stimulation of a protective immuneresponse is desirable, which may be accomplished via the methods of thisinvention, or utilizing the NK T cells, vaccines or compositions of thepresent invention include: Retroviridae (e.g., human immunodeficiencyviruses, such as HIV-1 (also referred to as HTLV-III, LAV orHTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP;Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses,human coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g.,strains that cause gastroenteritis); Togaviridae (e.g., equineencephalitis viruses, rubella viruses); Flaviridae (e.g., dengueviruses, encephalitis viruses, yellow fever viruses); Coronaviridae(e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitisviruses, rabies viruses); Filoviridae (e.g., ebola viruses);Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measlesvirus, respiratory syncytial virus); Orthomyxoviridae (e.g. influenzaviruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses,phleboviruses and Nairo viruses); Arena viridae (hemorrhagic feverviruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses);Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesviruses'); Poxviridae (variola viruses, vaccinia viruses, pox viruses);and Iridoviridae (e.g. African swine fever virus); and unclassifiedviruses (e.g., the etiological agents of Spongiform encephalopathies,the agent of delta hepatities (thought to be a defective satellite ofhepatitis B virus), the agents of non-A, non-B hepatitis (class1=internally transmitted; class 2=parenterally transmitted (i.e.,Hepatitis C); Norwalk and related viruses, and astroviruses).

Examples of infectious bacteria to which stimulation of a protectiveimmune response is desirable, which may be accomplished via the methodsof this invention, or utilizing the NK T cells, vaccines or compositionsof the present invention include: Helicobacter pylori, Borelliaburgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g. M.tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae),Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis,Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus),Streptococcus agalactiae (Group B Streptococcus), Streptococcus(viridans group), Streptococcus faecalis, Streptococcus bovis,Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenicCampylobacter sp., Enterococcus sp., Chlamidia sp., Haemophilusinfluenzae, Bacillus antracis, corynebacterium diphtheriae,corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridiumperfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasturella multocida, Bacteroides sp., Fusobacteriumnucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponemapertenue, Leptospira, Actinomyces israelli and Francisella tularensis.

Examples of infectious fungi to which stimulation of a protective immuneresponse is desirable, which may be accomplished via the methods of thisinvention, or utilizing the NK T cells, vaccines or compositions of thepresent invention include: Cryptococcus neoformans, Histoplasmacapsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydiatrachomatis, Candida albicans. Other infectious organisms (i.e.,protists) include: Plasmodium sp., Leishmania sp., Schistosoma sp. andToxoplasma sp.

It is to be understood that the modulation of any immune response, viathe use of the NK T cell populations, vaccines or compositions of thisinvention are to be considered as part of this invention, and anembodiment thereof.

In another embodiment, the NK T cell populations of this invention maybe isolated, culture-expanded, or otherwise manipulated, as will beunderstood by one skilled in the art. In one embodiment, the NK T cellsas derived by the methods of this invention, may be further engineeredto express substances of interest. In one embodiment, the NK T cellpopulations may be engineered to express particular adhesion molecules,or other targeting molecules, which, when the cells are provided to asubject, facilitate targeting of the NK T cell populations to a site ofinterest. For example, when NK T cell activity is desired to modulate animmune response at a mucosal surface, the isolated NK T cell populationsof this invention may be further engineered to express the α_(c)β₇adhesion molecule, which has been shown to play a role in mucosalhoming. The cells can be engineered to express other targetingmolecules, such as, for example, an antibody specific for a proteinexpressed at a particular site in a tissue, or, in another embodiment,expressed on a particular cell located at a site of interest, etc.Numerous methods are well known in the art for engineering the cells,and may comprise the use of a vector, or naked DNA, wherein a nucleicacid coding for the targeting molecule of interest is introduced via anynumber of methods well described.

A nucleic acid sequence of interest may be subcloned within a particularvector, depending upon the desired method of introduction of thesequence within cells. Once the nucleic acid segment is subcloned into aparticular vector it thereby becomes a recombinant vector.Polynucleotide segments encoding sequences of interest can be ligatedinto commercially available expression vector systems suitable fortransducing/transforming mammalian cells and for directing theexpression of recombinant products within the transduced cells. It willbe appreciated that such commercially available vector systems caneasily be modified via commonly used recombinant techniques in order toreplace, duplicate or mutate existing promoter or enhancer sequencesand/or introduce any additional polynucleotide sequences such as forexample, sequences encoding additional selection markers or sequencesencoding reporter polypeptides.

There are a number of techniques known in the art for introducing theabove described recombinant vectors into cells, such as, but not limitedto: direct DNA uptake techniques, and virus, plasmid, linear DNA orliposome mediated transduction, receptor-mediated uptake andmagnetoporation methods employing calcium-phosphate mediated andDEAE-dextran mediated methods of introduction, electroporation,liposome-mediated transfection, direct injection, and receptor-mediateduptake (for further detail see, for example, “Methods in Enzymology”Vol. 1-317, Academic Press, Current Protocols in Molecular Biology,Ausubel F. M. et al. (eds.) Greene Publishing Associates, (1989) and inMolecular Cloning: A Laboratory Manual, 2nd Edition, Sambrook et al.Cold Spring Harbor Laboratory Press, (1989), or other standardlaboratory manuals). Bombardment with nucleic acid coated particles isalso envisaged.

The efficacy of a particular expression vector system and method ofintroducing nucleic acid into a cell can be assessed by standardapproaches routinely used in the art. For example, DNA introduced into acell can be detected by a filter hybridization technique (e.g., Southernblotting) and RNA produced by transcription of introduced DNA can bedetected, for example, by Northern blotting, RNase protection or reversetranscriptase-polymerase chain reaction (RT-PCR). The gene product canbe detected by an appropriate assay, for example by immunologicaldetection of a produced protein, such as with a specific antibody, or bya functional assay to detect a functional activity of the gene product,such as an enzymatic assay. If the gene product of interest to beexpressed by a cell is not readily assayable, an expression system canfirst be optimized using a reporter gene linked to the regulatoryelements and vector to be used. The reporter gene encodes a geneproduct, which is easily detectable and, thus, can be used to evaluateefficacy of the system. Standard reporter genes used in the art includegenes encoding β-galactosidase, chloramphenicol acetyl transferase,luciferase and human growth hormone, or any of the marker proteinslisted herein.

In another embodiment, this invention provides a method for producing anisolated, culture-expanded NK T cell population, comprising contactingVα14i, or Vα24i T cells with dendritic cells and a compound of thisinvention, for a period of time resulting in antigen-specific T cellexpansion and isolating the expanded T cells thus obtained, therebyproducing an isolated, culture-expanded NK T cell population.

In one embodiment, the method for producing an isolated culture-expandedNK T cell population, further comprises the step of adding a cytokine orgrowth factor to the dendritic cell, NK T cell culture. In oneembodiment, NK T cells secretion of interleukin-2, interferon-γ orinterleukin-4 is detected, at which time the NK T cells are used in themethods of this invention.

Dendritic cells stimulated NK T cell cytokine production, whenpresenting a compound of this invention, in the context of CD1. Inanother embodiment of this invention, the stimulated NK T cells mayinduce maturation of the dendritic cells, which may be mediated via TCRand CD1d/glycolipid interactions, and engagement of the CD40/CD40Linteraction. This in turn, in another embodiment, may promote IL-12secretion by the dendritic cells, and/or upregulation of, inter-alia,MHC molecules, DEC-205, or costimulatory molecules such as the B7family. Dendritic cell maturation as a result of this interaction, may,in another embodiment, lead to enhanced adaptive immune responses, whichin another embodiment, includes adjuvant activity of the compounds ofthis invention.

In one embodiment, the term “dendritic cell” (DC) refers toantigen-presenting cells, which are capable of presenting antigen to Tcells, in the context of CD1. In one embodiment, the dendritic cellsutilized in the methods of this invention may be of any of several DCsubsets, which differentiate from, in one embodiment, lymphoid or, inanother embodiment, myeloid bone marrow progenitors. In one embodiment,DC development may be stimulated via the use of granulocyte-macrophagecolony-stimulating-factor (GM-CSF), or in another embodiment,interleukin (IL)-3, which may, in another embodiment, enhance DCsurvival.

In another embodiment, DCs may be generated from proliferatingprogenitors isolated from bone marrow. In another embodiment, DCs may beisolated from CD34+ progenitors as described by Caux and Banchereau inNature in 1992, or from monocytes, as described by Romani et al, J. Exp.Med. 180: 83-93 '94 and Bender et al, J. Immunol. Methods, 196: 121-135,'96 1996. In another embodiment, the DCs are isolated from blood, asdescribed for example, in O'Doherty et al, J. Exp. Med. 178: 1067-10781993 and Immunology 82: 487-493 1994, all methods of which areincorporated fully herewith by reference.

In one embodiment, the DCs utilized in the methods of this invention mayexpress myeloid markers, such as, for example, CD11c or, in anotherembodiment, an IL-3 receptor-α (IL-3Rα) chain (CD123). In anotherembodiment, the DCs may produce type I interferons (IFNs). In oneembodiment, the DCs utilized in the methods of this invention expresscostimulatory molecules. In another embodiment, the DCs utilized in themethods of this invention may express additional adhesion molecules,which may, in one embodiment, serve as additional costimulatorymolecules, or in another embodiment, serve to target the DCs toparticular sites in vivo, when delivered via the methods of thisinvention, as described further hereinbelow.

In one embodiment, the DCs may be obtained from in vivo sources, suchas, for example, most solid tissues in the body, peripheral blood, lymphnodes, gut associated lymphoid tissue, spleen, thymus, skin, sites ofimmunologic lesions, e.g. synovial fluid, pancreas, cerebrospinal fluid,tumor samples, granulomatous tissue, or any other source where suchcells may be obtained. In one embodiment, the dendritic cells areobtained from human sources, which may be, in another embodiment, fromhuman fetal, neonatal, child, or adult sources. In another embodiment,the dendritic cells used in the methods of this invention may beobtained from animal sources, such as, for example, porcine or simian,or any other animal of interest. In another embodiment, dendritic cellsused in the methods of this invention may be obtained from subjects thatare normal, or in another embodiment, diseased, or in anotherembodiment, susceptible to a disease of interest.

Dendritic cell separation may accomplished in another embodiment, viaany of the separation methods as described herein. In one embodiment,positive and/or negative affinity based selections are conducted. In oneembodiment, positive selection is based on CD86 expression, and negativeselection is based on GR1 expression.

In another embodiment, the dendritic cells used in the methods of thisinvention may be generated in vitro by culturing monocytes in presenceof GM-CSF and IL-4.

In one embodiment, the dendritic cells used in the methods of thisinvention may express CD83, an endocytic receptor to increase uptake ofthe autoantigen such as DEC-205/CD205 in one embodiment, or DC-LAMP(CD208) cell surface markers, or, in another embodiment, varying levelsof the antigen presenting MHC class I and II products, or in anotherembodiment, accessory (adhesion and co-stimulatory) molecules includingCD40, CD54, CD58 or CD86, or any combination thereof. In anotherembodiment, the dendritic cells may express varying levels of CD115,CD14, CD68 or CD32.

In one embodiment, mature dendritic cells are used for the methods ofthis invention. In one embodiment, the term “mature dendritic cells”refers to a population of dendritic cells with diminished CD115, CD14,CD68 or CD32 expression, or in another embodiment, a population of cellswith enhanced CD86 expression, or a combination thereof. In anotherembodiment, mature dendritic cells will exhibit increased expression ofone or more of p55, CD83, CD40 or CD86 or a combination thereof. Inanother embodiment, the dendritic cells used in the methods of thisinvention will express the DEC-205 receptor on their surface. In anotherembodiment, maturation of the DCs may be accomplished via, for example,CD40 ligation, CpG oligodeoxyribonucleotide addition, ligation of theIL-1, TNFα or TOLL like receptor ligand, bacterial lipoglycan orpolysaccharide addition or activation of an intracellular pathway suchas TRAF-6 or NF-κB.

In one embodiment, inducing DC maturation may be in combination withendocytic receptor delivery of a preselected antigen. In one embodiment,endocytic receptor delivery of antigen may be via the use of the DEC-205receptor.

In one embodiment, the maturation status of the dendritic may beconfirmed, for example, by detecting either one or more of 1) anincrease expression of one or more of p55, CD83, CD40 or CD86 antigens;2) loss of CD115, CD14, CD32 or CD68 antigen; or 3) reversion to amacrophage phenotype characterized by increased adhesion and loss ofveils following the removal of cytokines which promote maturation ofPBMCs to the immature dendritic cells, by methods well known in the art,such as, for example, immunohistochemistry, FACS analysis, and others.

In one embodiment, the dendritic cells used for the methods of thisinvention may express, or in another embodiment, may be engineered toexpress a costimulatory molecule. In one embodiment, dendritic cellsused for the methods of this invention are enriched for CD86^(high) orCD80^(high) expression.

In another embodiment, the dendritic cells used in the methods of thisinvention are selected for their capacity to expand antigen-specific NKT cells. In one embodiment, the DCs are isolated from progenitors orfrom blood for this purpose. In another embodiment, dendritic cellsexpressing high amounts of DEC-205/CD205 are used for this purpose.

NK T cell expansion, in one embodiment, is antigen-specific. In oneembodiment, a compound of this invention is supplied in the culturesimultaneously with dendritic cell contact with the NK T cells. Inanother embodiment, dendritic cells, which have already processedantigen are contacted with the NK T cells.

In one embodiment, the term “contacting a target cell” refers herein toboth direct and indirect exposure of cell to the indicated item. In oneembodiment, contact of NK T cells with a compound of this invention, acytokine, growth factor, dendritic cell, or combination thereof, isdirect or indirect. In one embodiment, contacting a cell may comprisedirect injection of the cell through any means well known in the art,such as microinjection. It is also envisaged, in another embodiment,that supply to the cell is indirect, such as via provision in a culturemedium that surrounds the cell, or administration to a subject, via anyroute well known in the art, and as described hereinbelow.

Methods for priming dendritic cells with antigen are well known to oneskilled in the art, and may be effected, as described for example Hsu etal., Nature Med. 2:52-58 (1996); or Steinman et al. Internationalapplication PCT/US93/03141.

In one embodiment, a compound of this invention is added to a culture ofdendritic cells prior to contact of the dendritic cells with NK T cells.In one embodiment, a compound of this invention is used at aconcentration of between about 0.1 to about 200 μg/ml. In oneembodiment, 10-50 μg/ml is used. The dendritic cells are, in oneembodiment, cultured in the presence of the antigen for a sufficienttime to allow for uptake and presentation, prior to, or in anotherembodiment, concurrent with culture with NK T cells. In anotherembodiment, the compound is administered to the subject, and, in anotherembodiment, is targeted to the dendritic cell, wherein uptake occurs invivo, for methods as described hereinbelow.

Antigenic uptake and processing, in one embodiment, can occur within 24hours, or in another embodiment, longer periods of time may benecessary, such as, for example, up to and including 4 days or, inanother embodiment, shorter periods of time may be necessary, such as,for example, about 1-2 hour periods.

In one embodiment, NK T cells may be cultured with dendritic cells witha dendritic cell to T cell ratio of 10:1 to 1:1 to 1:10, which ratio, insome embodiments is dependent upon the purity of the NKT cell populationused. In one embodiment, about 20,000-100,000 cells/well (96-well flatbottom plate) of a NKT cell line, or 5 million per ml T cells, or inanother embodiment, 200,000-400,000 cells/well of enriched NKT areadministered to a subject, for some of the methods of this invention.

In one embodiment, about 5 million T cells are administered to asubject, for some of the methods of this invention.

In another embodiment, the NK T cells expanded by the dendritic cells inthe methods of this invention are autologous, syngeneic or allogeneic,with respect to the dendritic cells.

In another embodiment, the dendritic cells used in the methods of thisinvention are isolated from a subject suffering from an autoimmunedisease or disorder, cancer, an infection, which in one embodiment, isHIV, mycobacterial or malarial infection.

In another embodiment, the dendritic cells used in the methods of thisinvention are isolated from a subject with an inappropriate orundesirable immune response, or in another embodiment, the dendriticcells used in the methods of this invention are isolated from a subjectwith an allergic response.

In one embodiment, the NK T cells can be used to modulate an immuneresponse, in a disease-specific manner. It is to be understood that anyimmune response, wherein it is desired to enhance cytokine production,or elicit a particular cytokine profile, including interferon-γ,interleukin-2 and/or interleukin-4, the NK T cells of this invention maybe thus utilized, and represents an embodiment of this invention.

In another embodiment, the methods of this invention may furthercomprise the step of culturing previously isolated, NK T cells withadditional dendritic cells, and a compound of this invention, for aperiod of time resulting in further NK T cell expansion, cytokineproduction, or a combination thereof.

In another embodiment, this invention provides a method for delayingonset, reducing incidence or suppressing a disease in a subject,comprising the steps of contacting in a culture NK T cells withdendritic cells and a compound of this invention, for a period of timeresulting in NK T cell expansion, cytokine production or a combinationthereof, and administering NK T cells thus obtained to the subject,wherein the NK T cells delay onset, reduce incidence or suppress adisease in the subject, thereby delaying onset, reducing incidence orsuppressing a disease in the subject.

In one embodiment, cells for administration to a subject in thisinvention may be provided in a composition. These compositions may, inone embodiment, be administered parenterally or intravenously. Thecompositions for administration may be, in one embodiment, sterilesolutions, or in other embodiments, aqueous or non-aqueous, suspensionsor emulsions. In one embodiment, the compositions may comprise propyleneglycol, polyethylene glycol, injectable organic esters, for exampleethyl oleate, or cyclodextrins. In another embodiment, compositions mayalso comprise wetting, emulsifying and/or dispersing agents. In anotherembodiment, the compositions may also comprise sterile water or anyother sterile injectable medium. In another embodiment, the compositionsmay comprise adjuvants, which are well known to a person skilled in theart (for example, vitamin C, antioxidant agents, etc.) for some of themethods as described herein, wherein stimulation of an immune responseis desired, as described further hereinbelow.

In one embodiment, the compounds, cells, vaccines or compositions ofthis invention may be administered to a subject via injection. In oneembodiment, injection may be via any means known in the art, and mayinclude, for example, intra-lymphoidal, or subcutaneous injection.

In another embodiment, the NK T cells and dendritic cells foradministration in this invention may express adhesion molecules fortargeting to particular sites. In one embodiment, NK T cells and/ordendritic cells may be engineered to express desired molecules, or, inanother embodiment, may be stimulated to express the same. In oneembodiment, the DC cells for administration in this invention mayfurther express chemokine receptors, in addition to adhesion molecules,and in another embodiment, expression of the same may serve to attractthe DC to secondary lymphoid organs for priming. In another embodiment,targeting of DCs to these sites may be accomplished via injecting theDCs directly to secondary lymphoid organs through intralymphatic orintranodal injection.

In one embodiment, the antigen is delivered to dendritic cells in vivoin the steady state, which, in another embodiment, leads to expansion ofdisease specific NK T cells. Antigen delivery in the steady state can beaccomplished, in one embodiment, as described (Bonifaz, et al. (2002)Journal of Experimental Medicine 196: 1627-1638; Manavalan et al. (2003)Transpl Immunol. 11: 245-58).

In another embodiment, select types of dendritic cells in vivo functionto prime the NK T cells.

In one embodiment, this invention provides a method for modulating animmune response, which is an inappropriate or undesirable response. Inone embodiment, the immune response is marked by a cytokine profilewhich is deleterious to the host.

In one embodiment, the NK T cells of this invention may be administeredto a recipient contemporaneously with treatment for a particulardisease, such as, for example, contemporaneous with standard anti-cancertherapy, to serve as adjunct treatment for a given cancer. In anotherembodiment, the NK T cells of this invention may be administered priorto the administration of the other treatment.

In another embodiment, this invention provides a method for modulatingan immune response, which is directed to infection with a pathogen, andthe immune response is not protective to the subject.

In one embodiment, the pathogen may mimic the subject, and initiate anautoimmune response. In another embodiment, infection with the pathogenresults in inflammation, which damages the host. In one embodiment, theresponse results in inflammatory bowel disease, or in anotherembodiment, gastritis, which may be a result, in another embodiment, ofH. pylori infection.

In another embodiment, the immune response results in a cytokineprofile, which is not beneficial to the host. In one embodiment, thecytokine profile exacerbates disease. In one embodiment, a Th2 responseis initiated when a Th1 response is beneficial to the host, such as forexample, in lepromatous leprosy. In another embodiment, a Th1 responseis initiated, and persists in the subject, such as for example,responses to the egg antigen is schistosomiasis.

According to this aspect, and in one embodiment, administration of theNK T cells alters the immune response initiated in the subject, was notbeneficial to the subject. In another embodiment, the method may furthercomprise the step of administering an agent to the subject, which iffurther associated with protection from the pathogen.

In one embodiment, the term “modulating” refers to initiation,augmentation, prolongation, inhibition, suppression or prevention of aparticular immune response, as is desired in a particular situation. Inone embodiment, modulating results in diminished cytokine expression,which provides for diminished immune responses, or their prevention. Inanother embodiment, modulation results in the production of specificcytokines which have a suppressive activity on immune responses, or, inanother embodiment, inflammatory responses in particular. In anotherembodiment, modulating results in enhanced cytokine expression, whichprovides for enhanced immune responses, or their stimulation. In anotherembodiment, modulation results in the production of specific cytokineswhich have a stimulatory activity on immune responses, or, in anotherembodiment, responses to infection, or neoplasia, in particular.

In one embodiment, this invention provides a method for modulating animmune response in a subject, comprising the steps of contacting adendritic cell population in vivo with compound of this invention,whereby the dendritic cell population contacts NK T cells in thesubject, wherein NK T cell contact promotes cytokine production from theNK T cell population, thereby modulating an immune response in asubject.

In one embodiment, the term “modulating” refers to stimulating,enhancing or altering the immune response. In one embodiment, the term“enhancing an immune response” refers to any improvement in an immuneresponse that has already been mounted by a mammal. In anotherembodiment, the term “stimulating an immune response” refers to theinitiation of an immune response against an antigen of interest in amammal in which an immune response against the antigen of interest hasnot already been initiated. It is to be understood that reference tomodulation of the immune response may, in another embodiment, involveboth the humoral and cell-mediated arms of the immune system, which isaccompanied by the presence of Th2 and Th1 T helper cells, respectively,or in another embodiment, each arm individually. For further discussionof immune responses, see, e.g., Abbas et al. Cellular and MolecularImmunology, 3rd Ed., W. B. Saunders Co., Philadelphia, Pa. (1997).

Modulation of an immune response can be determined, in one embodiment,by measuring changes or enhancements in production of specific cytokinesand/or chemokines for either or both arms of the immune system. In oneembodiment, modulation of the immune response resulting in thestimulation or enhancement of the humoral immune response, may bereflected by an increase in IL-6, which can be determined by any numberof means well known in the art, such as, for example, by ELISA or RIA.In another embodiment, modulation of the immune response resulting inthe stimulation or enhancement of the cell-mediated immune response, maybe reflected by an increase in IFN-γ or IL-12, or both, which may besimilarly determined.

In one embodiment, stimulating, enhancing or altering the immuneresponse is associated with a change in cytokine profile. In anotherembodiment stimulating, enhancing or altering the immune response isassociated with a change in cytokine expression. Such changes may bereadily measured by any number of means well known in the art, includingas described herein, ELISA, RIA, Western Blot analysis, Northern blotanalysis, PCR analysis, RNase protection assays, and others.

In one embodiment, the infection is a latent infection.

In another embodiment, the immune response inhibits disease progressionin the subject, or in another embodiment, the immune response inhibitsor prevents neoplastic transformation in the subject.

In one embodiment, inhibition or prevention of neoplastic transformationaccording to the methods of this invention may be effected via the useof tumor specific antigens, in addition to the compounds of thisinvention. In one embodiment, a tumor specific antigen may be, forexample, mutated proteins which are expressed as a result of aneoplastic, or preneoplastic events. In one embodiment, the antigen is amolecule associated with malignant tumor cells, such as, for examplealtered ras. Non-limiting examples of tumors for which tumor specificantigens have been identified include melanoma, B cell lymphoma, uterineor cervical cancer.

In one embodiment, a melanoma antigen such as the human melanomaspecific antigen gp75 antigen may be used, or, in another embodiment, incervical cancer, papilloma virus antigens may be used for the methods ofthis invention. Tumor specific idiotypic protein derived from B celllymphomas, or in another embodiment, antigenic peptide or protein isderived from the Epstein-Bass virus, which causes lymphomas may be used,as well.

In another embodiment, the antigenic peptide or protein is derived fromHER2/neu or chorio-embryonic antigen (CEA) for suppression/inhibition ofcancers of the breast, ovary, pancreas, colon, prostate, and lung, whichexpress these antigens. Similarly, mucin-type antigens such as MUC-1 canbe used against various carcinomas; the MAGE, BAGE, and Mart-1 antigenscan be used against melanomas. In one embodiment, the methods may betailored to a specific cancer patient, such that the choice of antigenicpeptide or protein is based on which antigen(s) are expressed in thepatient's cancer cells, which may be predetermined by, in otherembodiments, surgical biopsy or blood cell sample followed byimmunohistochemistry.

In one embodiment, the subject being treated via a method of thisinvention has a precancerous precursor, and/or is at an elevated riskfor cancer. Such elements are well known in the art, and may compriseinappropriate expression of a given surface marker or oncoprotein, thepresence of hyperplastic cells, or the subject may have at least onefamily member afflicted with a given cancer, or have a lifestyleassociated with enhanced risk for the incidence of cancer, such as, forexample, exposure to radiation, certain viral infections, smokingtobacco products, and others, as will be appreciated by one skilled inthe art.

It is to be understood that any disease, disorder or condition, wherebysuch disease, disorder or condition may be positively affected by theproduction of a given cytokine profile, or in another embodiment, ispositively affected by the presence of NK T cells, and may be sopositively affected via a method of this invention, is to be consideredas part of this invention.

The following non-limiting examples may help to illustrate someembodiments of the invention.

EXAMPLES

A number of glycolipids were synthesized (FIG. 5) and tested them forNKT cell activation. These included glycolipids of bacterial origin(compounds 5, 6, 17, and 18), α-GalCer analogues modified on thegalactose moiety and acyl group, and variations of sulfatide, the onlyknown promiscuous ligand for CD1. The bacterial glycolipids includethose isolated from the outer membrane of Sphingomonas wittichii[Kawahara, K., Kubota, M., Sato, N., Tsuge, K. & Seto, Y. (2002) FEMSMicrobiol. Lett. 214, 289-294] and glycolipids from Borreliaburgdorferi, the Lyme disease spirochete. CD1d-deficient (CD1d ⁻/⁻) micewere shown to have impaired host resistance to infection by B.burgdorferi making its glycolipids attractive compounds for furtherstudy as possible natural CD1d antigens [Kumar, H., Belperron, A.,Barthold, S. W. & Bockenstedt, L. K. (2000) J. Immunol. 165, 4797-4801].The structures of its two major glycolipids were recently elucidated ascholesteryl 6-O-acyl-β-D-galactopyranoside 5 (B. burgdorferi glycolipid1, BbGL-I) and 1,2-di-O-acyl-3-O-α-D-galactopyranosyl-sn-glycerol 6(BbGL-II). The Sphingomonas glycolipids, two new α-linkedglycosphingolipids 5 and 6, (GSL-1 and GSL-2 respectively) differ mostsignificantly from α-GalCer in the carbohydrate moiety as they containgalactosyluronic acids as the polar head group [Ben-Menachem, G.,Kubler-Kielb, J., Coxon, B., Yergey, A. & Schneerson, R. (2003) Proc.Natl. Acad. Sci. USA 100, 7913-7918]. However, they are morephysiologically relevant as natural ligands for CD1d-mediated NKT-cellactivation since they originate from bacteria. Biological experimentsfurther show that galactouronic sphingolipids stimulate IL-2 secretionin 1.2 (Vα14 Vβ8.2 DN3A4) NKT cell hybridomas. An α-GalCer analogue 4,3-O-sulfo-galactosylceramide (3-O-sulfo-GalCer) also caused significantIL-2 secretion demonstrating that Vα14i NKT cell response is lesssensitive to modification at the 3-OH position of galactose. Bycontrast, any modification made at the 2-OH position of galactoseabolished all biological activity. Most other synthetic analogues,however, were active. In addition, reactivity of human Vα24i NKT cellsto GSL-1 and GSL-2 and sulfatides were conserved.

Example 1 Synthesis of analogues of glycolipid α-galactosyl ceramide:3-O-sulfo-α-galactosyl ceramide Preparation of Reagents

Reagents

All chemicals were purchased as reagent grade and used without furtherpurification. Dichloromethane (CH₂Cl₂, DCM) was distilled over calciumhydride and tetrahydrofuran (THF) over sodium/benzophenone. Anhydrousmethanol (MeOH) and pyridine (Py) were purchased from a commercialsource.

General Assay Information:

Reactions were monitored with analytical thin-layer chromatography (TLC)on silica gel 60 F₂₅₄ glass plates and visualized under UV (254 nm)and/or by staining with acidic ceric ammonium molybdate. Flash columnchromatography was performed on silica gel 60 Geduran (35-75 μm EMScience). ¹H NMR spectra were recorded on a 400-500- or 600-Hz NMRspectrometer at 20° C. Chemical shift (in ppm) was determined relativeto tetramethylsilane (δ 0 ppm) in deuterated solvents. Couplingconstant(s) in hertz (Hz) were measured from one-dimensional spectra.¹³C Attatched Proton Test (C-Apt) spectra were obtained with theNMR-400, 500 or 600 spectrometer (100, 125 or 150 Hz) and werecalibrated with either CDCl₃ (δ 77.23 ppm) or Py-d₅ (δ 123.87 ppm).

p-Methylphenyl2-O-benzyl-4,6-O-benzylidene-3-O-levulinyl-1-thio-D-galactopyranoside(II)

3 grams of I (6.45 mmol) was dissolved in DCM. LevOH (0.9 ml, 1.35 eq),EDC (1.6 g, 1.3 eq) and DMAP (197 mg, 0.25 eq) were added. The reactionwas allowed to proceed overnight while covered in foil. The reaction wasthen diluted with DCM, washed with water, saturated sodium bicarbonatesolution, brine and dried over sodium sulfate. After removal of thesolvent the mixture was purified by column chromatography(Hexanes:EtOAc:DCM 3:1:1) to give 2.83 g of II in 78% yield.

¹H (CDCl₃ 500 MHz) δ=7.61-7.03 (m, 14H), 5.48 (s, 1H), 4.98 (dd, J=3.7Hz, J=9.6 Hz, 1H), 4.77 (d, J=11.0 Hz, 1H), 4.63 (d, J=9.5 Hz, 1H), 4.51(d, J=11 Hz, 1H), 4.36-4.32 (m, 2H), 3.99-3.97 (m, 1H), 3.90-3.86 (m,1H), 3.51 (s, 1H), 2.56-2.50 (m, 2H), 2.46-2.40 (m, 2H), 2.31 (s, 3H),2.09, (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ=206.05, 172.09, 138.18,137.76, 137.64, 133.11, 129.61, 128.98, 128.57, 128.22, 128.01, 127.68,127.57, 126.45, 100.83, 86.53, 75.41, 75.05, 73.77, 73.71, 69.09, 37.70,29.60, 27.99; HRMS (MALDI-FTMS) calcd. for C₃₂H₃₄O₇SNa [M+Na]⁺ 585.1923,found 585.1900.

2-O-benzyl-4,6-O-benzylidene-3-O-levulinyl-D-galactopyranoside (III)

II (600 mg, 1.07 mmol) was dissolved in 50 mL of acetone. The reactionmixture was cooled to 0° C., and NBS (228 mg, 1.28 mmol, 1.2 equiv) wasadded. The reaction mixture turned orange immediately. After 10 min thereaction was quenched by addition of solid NH₄Cl. The mixture wasdiluted with water and ethyl acetate, and the aqueous layer wasextracted with ethyl acetate (3×). The combined organic layer wasextracted with brine, dried over sodium sulfate, and evaporated. Theresidue was subjected to column chromatography (hexanes:ethyl EtOAc:DCM1:1:1) to give 442 mg (91%) of 7.

¹H (CDCl₃ 500 MHz) δ=7.50-7.25 (m, 10H), 5.48 (d, J=4.8, 1H), 5.38 (s,1H), 5.32 (dd, J=3.7 Hz, J=10.3 Hz, 1H), 4.94-4.90 (m, 1H), 4.73-4.62(m, 3H), 4.36 (d, J=3.3 Hz, 1H), 4.05 (dd, J=3.3 Hz, 10.3 Hz, 1H),4.00-3.98 (m, 2H), 3.93, (s, 1H), 3.52-3.51 (m, 1H), 2.71-2.53 (m, 4H),2.08 (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ=206.43, 177.73, 172.35, 172.24,138.41, 137.78, 137.63, 137.57, 128.89, 128.85, 128.38, 128.21, 128.03,127.75, 127.67, 127.51, 126.15, 126.12, 100.61, 97.50, 91.98, 77.57,74.68, 74.10, 73.82, 73.56, 73.38, 73.28, 70.55, 69.17, 68.93, 66.24,62.18, 37.82, 37.79, 29.67, 289.38, 28.11, 28.04; HRMS (MALDI-FTMS)calcd. for C₂₅H₂₉O₈ [M+H]⁺ 457.1862 found 457.1856.

O-(2-O-benzyl-4,6-O-benzylidene-3-O-levulinyl-D-galactopyranosyl)Trichloroacetimidate(IV)

To a solution of III (188.5 mg, 0.46 mmol) dissolved in 4 ml of DCM wasadded CCl₃CN (0.46 ml, 4.62 mmol) and DBU (31 μl, 0.21 mmol). After 2hours at room temperature, the dark solution was concentrated and thenpurified by flash chromatography Hexanes:EtOAc (2:1) and 1%triethylamine to yield 8 (211 mg, 77%).

¹H (CDCl₃ 500 MHz) δ=7.59-7.34 (m, 10H), 5.61 (s, 1H), 5.45 (dd, J=3.2Hz, 10.7 Hz, 1H), 4.80-4.72 (m, 2H), 4.60 (d, J=3.3 Hz, 2H), 4.38-4.33(m, 2H), 4.13-4.10 (dd, J=1.8 Hz, 12.5 Hz, 1H), 4.05 (s, 1H), 2.79-2.72(m, 2H), 2.65 (m, 2H), 2.16 (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ=206.43,177.73, 172.35, 172.27, 138.41, 137.78, 137.63, 137.57, 128.89, 128.85,128.38, 128.21, 128.03, 127.86, 127.75, 127.67, 127.51, 126.15, 126.12,100.61, 97.50, 91.98, 77.57, 74.68, 74.10, 73.56, 73.38, 73.28, 70.55,69.17, 68.93, 66.24, 6218, 37.82 37.79, 29.67, 29.38, 28.11, 28.04.

2-Azido-3,4-di-O-benzyl-1-O-(2-O-benzyl-4,6-O-benzylidene-3-O-levulinyl-α-D-galactopyranosyl)-D-ribo-octadeca-6-ene-1-ol(VI)

A solution of trichloroacetimidate IV (150 mg, 0.25 mmol, 1.5 equiv))and sphingosine derivative V (86 mg, 0.16 mmol) in 2.5 mL of anhydrousTHF was added over freshly dried powdered AW-300 molecular sieves andcooled to −20° C. TMSOTf (23 μL, 0.8 equiv) was slowly added to thesolution, and the mixture was warmed up to 0° C. in 2.5 hours. Thereaction was quenched by addition of Et₃N (0.1 mL), and the mixture wasdiluted with EtOAc and filtered through Celite. The organic layer waswashed with saturated aqueous NaHCO₃ and brine, dried (Na₂SO₄), andconcentrated. The residue was purified by column chromatography onsilica gel (hexanes:EtOAc 6:1) to furnish VI (57 mg, 46% based onconsumed acceptor V) as a syrup, and recover V (18 mg).

¹H NMR (CDCl₃, 400 MHz): δ=7.49-7.23 (m 20H), 5.56-5.45 (m 3H), 5.32(dd, 1H, J=3.5 Hz, 10.5 Hz), 4.98 (d, 1H, J=3.1 Hz), 4.70-4.51 (m, 6H),4.38 (m, 1H), 4.13-3.82 (m, 5H), 3.71-3.62 (m, 4H), 2.75-2.40 (m, 6H),2.08 (s, 3H), 2.06-1.97 (m, 2H), 1.25 (bs, 18H), 0.88 (t, 3H, J=7.0 Hz);¹³C NMR (125 MHz, CDCl₃) δ=206.30, 172.25, 138.21, 137.93, 137.67,132.60, 128.89, 128.37, 128.35, 128.33, 129.29, 128.08, 127.27, 128.08,127.78, 127.73, 127.69, 127.63, 127.60, 127.17, 124.69, 100.65, 98.61,79.41, 78.95, 74.06, 73.65, 73.41, 73.10, 71.94, 70.79, 69.02, 68.21,62.41, 61.97, 37.93, 31.89, 29.71-29.32, 28.19, 27.58, 22.66, 14.10;ESI-MS (positive-ion mode): m/z 982.4 [M+Na]⁺.

3,4-Di-O-benzyl-1-O-(2-O-benzyl-4,6-O-benzylidene-α-D-galactopyranosyl)-2-hexacosylamino-D-ribo-octadeca-6-ene-1-ol(X)

The azide VI (57 mg, 0.059 mmol) was dissolved in 2.0 mL of anhydrousTHF and cooled to 0° C. PMe₃ (0.4 mL of 1.0 M in toluene, 0.4 mmol) wasadded to the solution, and the reaction was warmed up to roomtemperature and stirred over night. After almost disappearance of thestarting material, 0.8 mL of aq 1 M NaOH was added to the mixture andstirred for 5 hours. CH₂Cl₂ was then added to the solution, and themixture was washed with brine, dried over Na₂SO₄, and concentrated. Theresidue was used for the next step without further purification.Hexacosanoic acid (35 mg, 0.088 mmol, 1.5 eq) was suspended in CH₂Cl₂(2.0 ml), and then DEPBT (26 mg 0.087 mmol, 1.5 eq) and DIEA (15 μL, 1.5eq) were added. The mixture was vigorously shaken for 1 h to give aclear light yellow solution in which above crude amine mixture VIIIa andVIIIb was added subsequently. The solution was stirred over night atroom temperature and then diluted with EtOAc and washed with saturatedNaHCO₃ and brine. The organic phase was dried over Na₂SO₄ andconcentrated to afford a solid (IXa and IXb, 57 mg), which was dissolvedin 2 mL Py-HOAc solution (3:1 v/v, contains 0.30M NH₂NH₂.HOAc) andstirred for 1.5 h at room temperature. After the usual workup similarlyas above, the residue was purified by column chromatography on silicagel (hexanes:EtOAc 2:1) to furnish X (40 mg, 56% over 3 steps) as asolid.

¹H NMR (CDCl₃, 400 MHz) δ=7.47-7.23 (m 20H), 5.67 (d, 1H, J=8.6 Hz),5.51-5.44 (m, 3H), 4.95 (d, 1H, J=2.7 Hz), 4.77-4.49 (m, 6H), 4.40 (m,1H), 4.21 (d, 1H, J=2.7 Hz), 4.12-4.07 (m, 2H), 3.94-3.58 (m, 8H), 2.45(m, 2H), 2.08-1.88 (m, 4H), 1.49 (m, 2H), 1.25 (bs, 62H), 0.88 (t, 6H,J=7.0 Hz); ¹³C NMR (CDCl₃, 100 MHz): δ=173.14, 138.50, 138.33, 137.74,132.57, 129.32, 128.62, 128.40, 128.08, 127.95, 127.85, 126.45, 125.20,101.37, 99.04, 79.97, 79.22, 76.29, 73.48, 73.41, 71.79, 69.50, 68.86,68.19, 62.94, 50.26, 36.96, 32.13, 29.91-29.56, 28.14, 27.78, 25.93,22.90, 14.34; HRMS (MALDI-FTMS) calcd for C₇₈H₁₁₉NO₉Na [M+Na]⁺1236.8777, found 1236.8741.

3,4-Di-O-benzyl-1-O-(2-O-benzyl-4,6-O-benzylidene-3-O-sulfo-α-D-galactopyranosyl)-2-hexacosylamino-D-ribo-octadeca-6-ene-1-ol,sodium salt (XI)

To a solution of X (40 mg, 0.033 mmol) in Py (2.5 mL) was added SO₃.Pycomplex (79 mg, 0.5 mmol, 15 eq). The mixture was stirred at roomtemperature for 2.5 hours. Water solution (2.5 mL) of NaHCO₃ (62 mg) wasadded to quench the reaction. The reaction mixture was diluted withCH₂Cl₂, and washed with brine, dried (Na₂SO₄), and concentrated. Theresidue was purified by column chromatography on silica gel (CH₂Cl₂:MeOH15:1) to give XI (39 mg, 90%) as a solid.

¹H NMR (CDCl₃/CD₃OD 1:1, 400 MHz) δ=7.87 (d, 1H, J=8.9 Hz), 7.58-7.17(m, 20H), 5.59 (s, 1H), 5.43 (m, 2H), 4.96 (m, 3H), 4.82 (m, 1H), 4.73(d, 1H, J=2.3 Hz), 4.62-4.58 (m, 2H), 4.52-4.44 (m, 2H), 4.19-3.99 (m,5H), 3.78 (bs, 2H), 3.66 (bs, 1H), 3.56 (m, 1H), 2.47 (m, 1H), 2.34 (m,1H), 2.13 (t, 2H, J=7.0 Hz), 2.01 (m, 2H), 1.54 (bs, 2H), 1.27 (bs,62H), 0.89 (t, 6H, J=7.0 Hz); ¹³C NMR (CDCl₃/CD₃OD 1:1, 100 MHz):δ=173.92, 138.30, 137.66, 137.57, 131.42, 128.43, 128.01-127.03, 125.95,125.66, 100.59, 98.99, 80.16, 79.75, 75.04, 74.84, 73.97, 73.60, 73.49,71.09, 68.74, 67.00, 62.70, 49.71, 49.62, 31.56, 29.29-29.02, 27.01,25.59, 22.27, 13.44; HRMS (MALDI-FTMS) calcd for C₇₈H₁₁₈NO₁₂SNaK [M+K]⁺1354.7909, found 1354.7933.

2-Hexacosylamino-1-O-(3-O-sulfo-α-D-galactopyranosyl)-D-ribo-1,3,4-octadecantriol,sodium salt (4)

XI (39 mg, 0.030 mmol) was dissolved in HOAc-MeOH (1:1 v/v, 6 mL). 80 mgof palladium black was added and the reaction solution was saturatedwith hydrogen by a balloon. After stirring at room temperature for 20hours, the catalyst was removed by filtration over Celite and washedwith CH₂Cl₂/MeOH (1:1) thoroughly. Evaporation of the solvent gave aresidue which was dissolved in CH₂Cl₂/MeOH (1:1) mixed solvent again andthen saturated NaHCO₃ (3 mL) was added to stir at room temperature forhalf an hour. After removal of the solvent, the residue was purified bycolumn chromatography on silica gel (CH₂Cl₂:MeOH 6:1) to give 4 (24 mg,83%) as a light yellow solid.

¹H NMR (CDCl₃/CD₃OD 1:1, 400 MHz) δ=4.95 (d, 1H, J=3.5 Hz), 4.49 (dd,1H, J=2.7 Hz, 10.2 Hz), 4.35 (m, 1H), 4.17 (m, 1H), 4.02 (dd, 1H, J=2.7Hz, 9.8 Hz), 3.88-3.85 (m, 2H), 3.80-3.72 (m, 4H), 3.69-3.65 (m, 2H),3.61-3.57 (m, 1H), 2.24 (t, 2H, J=7.4 Hz), 1.59 (m, 4H), 1.27 (bs, 68H),0.89 (t, 6H, J=7.0 Hz); ¹³C NMR (CDCl₃/CD₃OD 1:1, 100 MHz): δ=174.31,99.08, 77.57, 73.42, 71.64, 70.44, 67.81, 67.19, 66.48, 61.28, 49.90,35.89, 31.51, 31.32, 29.29-28.94, 25.53, 22.22, 13.34; HRMS (MALDI-FTMS)calcd for C₅₀H₉₈NO₁₂SNa₂ [M+Na]⁺ 982.6599, found 982.6585.

Synthesis Scheme

Sulfatide and α-galactosyl ceramide have similar structures and possessimmunostimulatory and immunomodulatory activity, when presented to Tcells via CD1. In order to determine whether hybrid molecules ofsulfatide and α-galactosyl ceramide, which are sulfate derivatives3-O-sulfo-α/β-galactosylceramides 10 and 4 (FIG. 1), have comparableactivity, the molecules were synthesized and evaluated forimmunostimulatory activity.

For the synthesis of 3-O-sulfo-a-galactosylceramides 4, selectivesulfation at 3″ OH of the galactose moiety is a key step. Typically,regioselective sulfation of the 3-hydroxyl of the sugar ring utilizesdibutylstannylene acetals as activated intermediates, however, thismethod can only be applied to β-galactosides; for α-galactosides, thedibutylstannylene acetal can form a complex between the 2-hydroxyl andthe anomeric oxygen to give the 2″-O-derivative by reaction with anelectrophile.

In order to address this, a 3″-lev and 2″-benzyl-4″,6″-benzylideneprotected trichloroacetimidate donor IV (FIG. 2). The temporaryprotecting group Lev, can be selectively removed after glycosylation inthe presence of hydrazine. The benzyl and benzylidene groups at 2,4,6positions direct the next α-glycosidic bond formation (Figueroa-Perez,S. et al Carbohydrate Res. 2000, 328, 95; Plettenburg, O. et al. J. Org.Chem. 2002, 67, 4559).

As shown in FIG. 2A, the preparation of IV started with the knownthioglycoside I in 50% yield over three steps. The sphingosine buildingblock V was employed in this synthesis, with donor IV coupled toacceptor V in the presence of TMSOTf, used as promoter to giveα-glycoside VI, in a moderate yield.

A staudinger reduction of VI with PMe₃, in NaOH solution was used tohydrolyze the imino-phosphorane intermediate VII, however, the Lev groupcannot survive under this conditions and approximate 50% of the Levgroup was cleaved to give an amine mixture of VIIIa and VIIIb (1:1)determined by ¹H NMR. Since VIIIa possesses a free C-3 hydroxyl, it iscrucial to choose a selective coupling reagent in the condensationbetween amine VIIIa and the fatty acid.

Since DEPBT [3-(diethoxyphosphoryloxy)-(1,2,3)-benzotriazin-4(3H)-one]can selectively form an amide bond in the presence of unprotectedhydroxyl groups, it was used in the reaction mixture with VIIIa, VIIIb,and hexacosanoic acid to give IXa and IXb, followed by deprotection ofthe remaining Lev groups using hydrazine to provide the desiredgalactosyl ceramide X in 56% yield over 3 steps. Treating the 3″-OH freeglycolipid X with Py.SO3 led to the sulfate derivative XI in high yield,which gave compound 4 upon hydrogenation with palladium black andneutralization with NaHCO₃ (aqueous solution) in 78% yield (FIG. 2B).

Example 2 Synthesis of analogues of glycolipid α-galactosyl ceramide3-O-sulfo-β-galactosylceramide

For the synthesis of 10, perbenzoylated trichloroacetimidate donor 40 isused in the glycosylation of the sphingosine acceptor V to yield aβ-galactosyl ceramide derivative XII (FIG. 3). After the Staudingerreduction of XII, a complex mixture was produced, with no isolation ofthe amine XV. Since the perbenzoylated galactosyl ceramide is sensitiveto basic conditions, a NaHSO₄ solution instead of a NaOH solution wasused for the reduction work-up procedure to decompose theimino-phosphorane intermediate XIV. However, hydrolyzation of XIV intoXV was very slow, and in turn, the longer reaction time led to thedegradation of glycosidic bond which attributed to complicated productformation.

Example 3 Synthesis of analogues of glycolipid α-galactosyl ceramide3-O-sulfo-β-galactosylceramide

Another synthetic strategy was used to synthesize3-O-sulfo-β-galactosylceramide. In this strategy, the azide was firstreduced, and the fatty acid coupled, prior to the glycosylation step(FIG. 4A). Compound XVIII was prepared from the sphingosine derivativeXVI (Plettenburg, O. et al. J. Org. Chem. 2002, 67, 4559) in 54% yieldover 2 steps.

Using TMSOTf as a promoter, the ceramide acceptor XVIII was reacted withdonor 40 to give the β-glycoside XIX in 54% yield. After debenzyolationand hydrogenation of XIX, the β-galactosyl ceramide XX was obtained inquantitative yield. XX was finally sulfated by Bu₂SnO/Me₃N.SO₃ andsubsequently neutralized by NaHCO₃ to give the product 10, in 80% yield(FIG. 4B) (Compostella, F et al. Tetrahedron 2002, 58, 8703).

Example 4 Recognition of Glycolipids by the Human NKT Cell Line Resultsin Cytokine Secretion Materials and Methods

Glycolipids

α-GalCer was obtained as described [Plettenburg, O., et al. (2002) J.Org. Chem. 67, 4559-64]. The intermediates 29, 36 and 40 (FIGS. 3 and4), were obtained as described [Plettenburg, O., et al. (2002) supra;Williams, L., et al. (1996) Tetrahedron 52, 11673-11694; Deng, S. Y., Bet al. (1999) J. Org. Chem. 64, 7265-7266]. The compounds 5, 6, 19, 30,33, 37, and 41 (FIGS. 3 and 4) were obtained as described hereinbelow.The remaining compounds, except 19, 10 and 4, and their intermediateswere obtained as described hereinabove.

Sphingosine Acceptor

The synthesis scheme for the sphingosine acceptor (30) is shown in FIG.6. Compound 29 (3.31 g, 13.5 mmol) (Williams, L., et al. supra) wasdissolved in 70 ml of dry THF. The solution was cooled to −40° C. andvinyl grignard solution (31 ml of a 1 M solution in THF) was added via adropping funnel over a period of 1 hr. The temperature was kept between−20° C. and −40° C. The reaction mixture was allowed to warm to roomtemperature and stirred for another hr. The reaction was quenched byaddition of 60 ml of saturated (NH₄)₂SO₄ solution and evaporated todryness. The residue was diluted with water and extracted with ethylacetate (3×). The combined organic layer was extracted with brine, driedover MgSO₄ and evaporated to give a yellow oil. Column chromatography(Hex:EtOAc 3:1) yielded the syn diastereomer (2.11 g, 8.2 mmol,anti/syn=3.5:1) in 61% yield. Then the syn diastereomer (300 mg, 1.16mmol) was dissolved in 1 ml of dry dichloromethane in a two-necked flaskequipped with a reflux condenser under argon. 486 mg (3.48 mmol) ofpentadecene was added via a syringe. A solution of 20 mg (2 mol %) ofGrubb's second generation catalyst (purchased from Strem Chemicals) in 1ml of dichloromethane was added and the solution was heated under rapidreflux for 40 hr. The reaction mixture was evaporated and then directlychormatographed (Hex:EtOAc 6:1) which yielded (0.82 mmol, 71%) of thedesired product.

Synthesis of Glycolipids

The synthesis scheme is shown in FIG. 4. A solution oftrichloroacetimidate 32 (160.4 mg, 0.258 mmol) and sphingosine acceptor31 (100 mg, 0.198 mmol) in 4 ml of anhydrous Et₂O and 2 ml of anhydrousTHF was added over freshly dried 4 Å molecular sieves and cooled to −50°C. Trimethylsilylmethyl trifluoromethanesulfonate (TMSOTf) (3.33 mg,0.0198 mmol) was added and the mixture stirred at −50° C. for 1 hour.The mixture was allowed to warm to −20° C. and another 3.33 mg of TMSOTfwas added. The mixture was then slowly allowed to warm to roomtemperature and stirred for 3 hour. The solution was then diluted withEtOAc and filtered over celite. The organic layer was washed withsaturated aqueous NaHCO₃ and brine, dried (MgSO₄), and concentrated. Theresidue was purified by column chromatography on silica gel(toluene:EtOAc 12:1) to give 128 mg (67.5%, 0.134 mmol) of 33.

Compound 34 (36 mg, 0.03 mmol), dissolved in 6 ml of EtOAc, was added to36 mg of 20 wt % palladium hydroxide in 1 ml of EtOAc and saturated withhydrogen. The reaction vessel was purged with hydrogen, and the mixturewas stirred at room temperature overnight. The reaction mixture wasfiltered and the solvent was evaporated. The above hydrogenated compoundwas dissolved in 2 ml THF, 1 ml water, and 1 ml methanol. LiOH (9 mg,0.14 mmol) was added to the solution and the reaction was stirred atroom temperature for four hours. 100 mg of Na₂CO₃ was added and themixture stirred for 30 minutes. The solvent was evaporated and theremaining residue was purified on silica gel by column chromatography(CH₂Cl₂:MeOH 4:1) to give 7.8 mg of 1 (38%, 0.0114 mmol, 2 steps).

After deprotection of compound 42 (14 mg, 0.017 mmol), Bu₂SnO (6.5 mg,0.0259 mmol) dissolved in 1 ml of MeOH was added. The mixture wasrefluxed under argon for 2 h. After evaporation of the solvent, Me₃N.SO₃(5 mg, 0.035 mmol) dissolved in 1 ml THF was added and the reaction wasallowed to proceed at room temperature for 6 hours. The solvent was thenremoved under reduce pressure and the residue dissolved in CHCl₃/MeOH1:1 (1 mL) and loaded onto an ion exchange column (Dowex 50X8 Na+ form).After elution with CHCl₃/MeOH 1:1, the mixture was concentrated andpurified by column chromatography (CH₂Cl₂:MeOH 5:1) to give 18 (14.4 mg,95%).

1.2 Hybridoma Assay

CD1d reactive T cell hybridomas with an invariant Va14i T cell antigenreceptor a chain were used, as described (Sidobre, S., et al. (2004)Proc. Natl. Acad. Sci. USA 101, 12254-12259). T cell hybridomas werestimulated with the indicated glycolipids that were added either toplates coated with soluble CD1d, or with CD transfected A20 B lymphomacells, as described (Elewaut, D., et al. (2003) J. Exp. Med. 198,1133-1146). As a measure of T cell activation, IL-2 release into thetissue culture medium was measured after 16 hours culture by an ELISAassay.

Generation of Vα24i Human NKT Cell Line

Human NKT cell lines, expressing the Vα24i T cell receptor as well asCD161, were generated as follows: Anti-CD161 monoclonal antibodies, andanti-CD14 monoclonal antibodies, each coupled to magnetic beads(Miltenyi biotec, Auburn, Calif.), were used sequentially to isolateCD161⁺ cells and CD14⁺ cells from leukopaks. Immature dendritic cellswere generated from the CD14⁺ cells after a two-day incubation in thepresence of 300 U/ml GM-CSF (R&D systems, Minneapolis, Minn.) and 100U/ml IL-4 (R&D systems, Minneapolis, Minn.). Following irradiation with2000 rads, the immature dendritic cells were co-cultured with syngeneicCD161⁺ cells in the presence of 100 ng/ml of alpha-galactosylceramideand 10 IU/ml of IL-2 (Invitrogen, Carlsbad Calif.) for 10 to 14 days.After stimulating the CD161⁺ cells a second time withalpha-galactosylceramide-pulsed, irradiated immature dendritic cells,NKT cell lines were shown by flow cytometry to express both CD161⁺ and V24i TCR (99% purity).

In Vitro Cytokine Secretion Assay Using Human NKT Cell Lines

IFN-γ and IL-4 secretion by the Vα24i human NKT cell line was determinedby ELISA (BD Pharmingen, San Diego, Calif.) after culture for 16 hours.For these assays, 1×10⁵ Vα24i human NKT cells were co-cultured with4×10⁵ irradiated, immature CD14⁺ dendritic cells, in the presence of theglycolipid compounds at 10 μg/ml in a 96-well flat-bottom plate.

Results

In order to test whether glycolipids of bacterial origin (5, 6, 8, 17)(represented in FIG. 5), or analogues thereof, which comprise structuressimilar to α-GalCer either at the sugar or lipid moiety, activate NKTcells through CD1d, the glycolipids were synthesized and assayed.Analogues 7 and 8 (FIG. 5) were prepared, and used to probe the effectof the carboxyl group on the sugar and the α-hydroxyl group on thelipid. Compounds 19, 10 and 4 contain a 3′-sulfate group with an α orβ-glycosidic linkage. 20-23 were prepared to probe the effect of the2′-modification of α-GalCer. Analogues of α-GalCer with modification ofthe lipid moiety were also prepared to probe their interaction with CD1dand the subsequent effect on NKT cell activation.

Mouse Vα14i NKT cells immortalized by cell fusion provided a convenientmethod for assaying the ability of the synthetic glycolipids to activateT cells. As shown in FIG. 8 a, the 3-O-sulfo-α-GalCer, 4, stimulatedsignificant IL-2 release from the hybridomas when used at 10 μg/ml. Doseresponse curves indicated, however, that this compound was somewhat lessactive than α-GalCer (data not shown) in this model. By contrast, everymodification of the 2 OH position of the galactose (compounds 10-13)that were tested abolished all biological activity. These data indicatethat the Vα14i NKT cell response to glycolipids apparently is moresensitive to modifications of the 2 than to the 3 position.

B. burgdorferi glycolipids (17-18) and compounds 24 and 25 weremoderately active in the 1.2 hybridoma assay. However IL-2 secretioncould only be detected when large quantities of the glycolipids wereused to stimulate the hybridoma cells.

CD1d coated plates were used to assay response of the hybridomas to theSphingomonas glycolipids (FIG. 8 b). A substantial level of IL-2secretion can be observed for all compounds. The structure of the sugarhead group significantly affected the activation of the hybridomas.α-GalCer and the galactose analogue 7, consistently solicited greaterIL-2 secretion when compared to the galacturonic acid derivatives. Alsoaffecting activity was the (S)-2-hydroxy of the fatty amide tail. Afully saturated tail was more greatly favored, suggesting that theα-hydroxyl group is not optimal. In fact the (S)-2-hydroxy appeared tohave a greater affect on activity as compound to compound 8, a galactoseanalogue, that was less able to activate IL-2 secretion when comparedwith 5, the galacturonic acid compound without the α-hydroxyl fattyamide. Though 7 and 8 are not known to be natural products, both couldbe precursors to compounds 5 and 6.

IFN-γ and IL-4 secretion from a Vα24i NKT cell line were assessed, afterstimulation with irradiated, syngeneic CD14⁺ immature dendritic cells inthe presence of 10 μg/ml of the glycolipids and 10 IU/ml of IL-2 (FIG. 9a). Stimulation of the NKT cell line by each glycolipid compoundresulted in significant IFN-γ and IL-4 secretion, when compared to thenegative control. While greater IFN-γ and IL-4 secretion was observedafter stimulation by the potent NKT cell agonist, α-GalCer, secretion ofIFN-γ and IL-4 by NKT cells stimulated by 1-10 μg/ml of3-O-sulfo-α-galactosylceramide was approximately half that of α-GalCer,but twice that induced by the other glycolipids. β-linked sulfatides 19and 10 were also observed to elicit both IFN-γ and IL-4 production. Infact, the level of cytokine secretion was comparable to the GSLs.

As illustrated in FIGS. 8C and 8D, interferon-γ secretion by human NKTcells in response to glycolipid presentation by CD14⁺ DCs, was superiorwhen the glycolipid was 3-sulfo-α-GalCer 4, as compared to α-GalCer, ata concentration of 10-20 μg/mL. Compound 4 efficiently stimulated IL-4and IFN-γ secretion, indicating that the modification of the 3″-OHposition of the galactose moiety with sulfate was useful in NKT cellsstimulation.

NKT cells activation was sensitive to the configuration of the anomericcarbon of glycolipid antigen molecules. 3-sulfo-β-GalCer 10 had minimalto no affinity for NKT lymphocytes due to the β-linkage of glycosidicbond, indicating that the α-linkage of the glycoside was essential forCD1 antigen binding.

Other α-GalCer analogues with an acetyl side chain or a shortenedbackbone were also tested and some activity was also observed (FIGS. 8Cand 8D).

Example 5 Human NKT Cell Lines Bind to Glycolipids in the Context ofCD1d Materials and Methods

In Vitro CD1d-Dimer Assay Using a Human NKT Cell Line

One mg of soluble divalent human CD1d-IgG1 fusion protein (humanCD1d-IgG1 dimers, BD Pharmingen) were incubated overnight with 10 M ofeach glycolipid at 32° C. and at neutral pH according to themanufacturer's protocol. The glycolipid-loaded CD1d-IgG1 dimers wereincubated with human NKT cells at 4° C. for 60 minutes, followed byincubation with PE-coupled anti-mouse IgG1 mAb (A85-1). The cells werealso surface stained with a PerCP-coupled anti-CD3 mAb (BD Pharmingen,San Diego, Calif.).

Results

Although glycolipids stimulated the NKT cell line, it does notnecessarily follow that the glycolipids were presented by CD1d moleculesand were capable of triggering the Vα24i T cell receptor expressed bythe NKT cells. Therefore, in order to demonstrate glycolipid antigenreactivity to the Vα24i T cell receptor at the single cell level, ahuman NKT cell line with human CD1d dimers loaded with differentglycolipids was stained, and unloaded CD1d dimers were used as anegative control. Each glycolipid-loaded dimer nearly universallystained the human NKT cells, while the unloaded dimer did not stainthese cells (FIG. 10).

Example 6 Computer Modeling of GSL Complexed to mCD1d Materials andMethods

Model Generation

A model of GSL 1 complexed with the crystal structure of mCD1d (Zeng,Z., et al. (1997) Science 277, 339-45) was generated by Autodock 3.0(Morris, G. M., et al. (1998) J. Comput. Chem. 19, 1639-1662).

Results

To further understand the interaction of bacterial glycolipid 1 withCD1d, a model of GSL 1 complexed with mCD1d was generated, and is shownin FIG. 11. According to the model, the fatty acyl chain extended intothe F′ pocket and the sphingosine chain toward the A′ pocket. Thisallowed for the polar head group to be oriented such that it was exposedfor recognition by a T cell antigen receptor. Numerous favorablecontacts could be observed between mCD1d and the glycosphingolipid.Among them, possible hydrogen bonding included interactions between thecarboxylate of the sugar and the backbone carbonyl of Asp80, and theamide nitrogen of the fatty acid tail with the Asp80 sidechain.

While it was thought that mCD1d to be somewhat accommodating in terms oflipid tail length on NKT cell reactivity, changes in the lipid length,composition, or addition of an α-hydroxyl group on the fatty acid, asseen in FIG. 11, could cause a slight shift in orientation of the sugarand thereby affect CD1d/glycolipid complex recognition by the T-cellreceptor. Substitution of galacturonic acid for galactose may producesimilar results. The perturbation caused by having the 6-OH oxidized toa carboxylic acid caused only moderate changes in NKT cell reactivity,thus the model provides an effective means for designing additionalligands.

Example 7 Synthesis of Analogues of Glycolipid α-Galactosyl Ceramide

A number of glycolipids were synthesized and tested for NKT cellactivation. A synthetic scheme is provided in scheme 1 below:

-   -   wherein R is selected from Table 1 below to provide the        corresponding compound.

TABLE 1 Compound No. R = 58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

110

111

Other compounds were synthesized as described in Xing G W et al. BioorgMed Chem. 2005 Apr. 15; 13(8):2907-16; and Wu D. et al., Proc Natl AcadSci USA. 2005 Feb. 1; 102(5): 1351-6.

All chemicals were purchased as reagent grade and used without furtherpurification. Dichloromethane (CH₂Cl₂) were distilled over calciumhydride. Tetrahydrofuran (THF) and ether were distilled over sodiummetal/benzophenone ketyl. Anhydrous N,N-dimethylformamide (DMF) waspurchased from Aldrich. Molecular sieves (MS) for glycosylation wereAW300 (Aldrich) and activated by flame. Reactions were monitored withanalytical thin layer chromatography (TLC) in EM silica gel 60 F254plates and visualized under UV (254 nm) and/or staining with acidicceric ammonium molybdate or ninhydrin. Flash column chromatography wasperformed on silica gel 60 Geduran (35-75 um, EM Science). ¹H NMRspectra were recorded on a Bruker DRX-500 (500 MHz) spectrometer or aBruker DRX-600 (600 MHz) spectrometer at 20° C. Chemical shifts (δ ppm)were assigned according to the internal standard signal oftetramethylsilane in CDCl₃ (δ=0 ppm). ¹³C NMR spectra were obtainedusing Attached Proton Test (APT) on a Bruker DRX-500 (125 MHz)spectrometer Bruker DRX-600 (150 MHz) spectrometer and were reported in6 ppm scale using the signal of CDCl₃ (δ=77.00 ppm) for calibration.Coupling constants (J) are reported in Hz. Splitting patterns aredescribed by using the following abbreviations: s, singlet; brs, broadsinglet; d, doublet; t, triplet; q, quartet; m, multiplet. ¹H NMRspectra are reported in this order: chemical shift; number(s) of proton;multiplicity; coupling constant(s).

To a stirred solution of Wittig reagent a (6.1 g, 11 mmol), preparedfrom 1-bromopentadecane and triphenylphosphine refluxed in toluene for 5days, in THF (30 mL) was added n-BuLi (1.6 mol/L in hexane, 6.4 mL, 10mmol) dropwise at −78° C., then the solution was stirred for 1 h at roomtemperature. After 1 h the solution was cooled to −78° C. and Garner'saldehyde A (2.1 g, 9.2 mmol) in THF (20 mL) was added. After stirringfor 1 h at room temperature, the solution was poured into ice-water andextracted with AcOEt. The organic layer was washed with brine, driedwith MgSO₄, and evaporated to dryness. The residue was purified by flashcolumn chromatography on silica gel (toluene 100%) to give B (2.6 g,66%) as a pale yellow oil. ¹H NMR (600 MHz, CDCl₃) δ 5.36-5.52 (2H, m),4.51-4.75 (1H, m), 4.05 (1H, dd, J=6.3 Hz, 8.6 Hz), 3.63 (1H, dd, J=3.3Hz, 8.6 Hz), 1.94-2.21 (2H, m), 1.20-1.66 (39H, m), 0.88 (1H, t, J=6.9Hz). ¹³C NMR (150 MHz, CDCl₃) δ 151.97, 131.98 (brs), 130.70 (brs),130.28 (brs), 129.36 (brs), 93.94 (brs), 93.38 (brs), 79.69 (brs),69.04, 54.55, 31.90, 29.72, 29.67, 29.65, 29.63, 29.59, 29.49, 29.33,29.29, 28.46, 22.66, 14.08. HRMS (ESI-TOF) for C₂₆H₄₉NO₃Na⁺ [M+Na]⁺calcd 446.3604, found 446.3602.

Synthesis of Compound C:

To a stirred solution of B (2.6 g, 6.0 mmol) and 1-methylmorpholineN-oxide (1.1 g, 9.0 mmol) in Bu^(t)OH and H₂O (1:1, 30 mL), OsO₄ (2.5w/v in Bu^(t)OH, 3.1 mL) was added at room temperature. The solution wasstirred overnight and quenched with Na₂SO₃ aq. The solution wasextracted 2 times with AcOEt, washed with brine, dried with MgSO₄, andevaporated to dryness. The residue was purified by flash columnchromatography on silica gel (CHCl₃ to CHCl₃:MeOH 20:1) to give C (1.6g, 56%) as a white solid. ¹H NMR (600 MHz, CDCl₃) δ 4.13-4.21 (2H, m),3.95-4.03 (1H, m), 3.55-3.65 (1H, m), 3.29-3.42 (2H, m), 1.38-1.68 (17H,m), 1.21-1.38 (24H, m), 0.88 (3H, t, J=7.1 Hz). ¹³C NMR (125 MHz, CDCl₃)δ: 153.93, 93.97, 81.35, 74.89, 73.85, 65.26, 59.41, 32.27, 31.87,29.65, 29.63, 29.59, 29.31, 28.31, 26.80, 26.18, 23.94, 22.64, 14.07.HRMS (ESI-TOF) for C₂₆H₅₁NO₅Na⁺ [M+Na]⁺ calcd 480.3659, found 480.3659.

Synthesis of Compound D:

To a stirred solution of C (328 mg, 0.72 mmol) and DMAP (cat.) inpyridine (5 mL) was added BzCl (0.20 mL, 1.8 mmol) and stirred at roomtemperature overnight. The solution was added Sat. NaHCO₃ aq., extractedwith AcOEt, washed with brine, dried with MgSO₄, and evaporated todryness. The residue was purified by flash column chromatography onsilica gel (Hex.:AcOEt 10:1) to give dibenzoylated product (471 mg) as acolorless oil. To a stirred solution of this compound in dry MeOH (5 mL)was added TFA (10 mL) dropwise at 0° C. After 2 h, the solution wasevaporated to dryness and co-evaporated with toluene 3 times. Theresidue was dissolved in dioxane (15 mL) and Sat. NaHCO₃ aq. (15 mL). Toa stirred solution, Na₂CO₃ (155 mg) and Boc₂O (320 mg, 1.5 mmol) wereadded and stirred overnight. This solution was extracted with AcOEt,washed brine, dried with MgSO₄ and evaporated to dryness. The residuewas purified by flash column chromatography on silica gel (Hex.:AcOEt5:1) to give D (301 mg, 67% over 3 steps) as a colorless oil. ¹H NMR(500 MHz, CDCl₃) δ: 8.05 (2H, d, J=7.2 Hz), 7.95 (2H, d, J=7.1 Hz), 7.63(1H, t, J=7.5 Hz), 7.46-7.54 (3H, m), 7.38 (2H, t, J=7.5 Hz), 5.50 (1H,d, J=9.6 Hz), 5.40 (1H, dd, J=2.4 Hz, 9.3 Hz), 5.33 (1H, d, J=9.5 Hz),4.00-4.07 (1H, m), 3.64-3.67 (2H, m), 2.55-2.65 (1H, m), 1.96-2.10 (2H,m), 1.48 (9H, s), 1.20-1.45 (24H, m), 0.88 (3H, t, J=7.0 Hz). ¹³C NMR(125 MHz, CDCl₃) δ: 167.03, 166.15, 155.54, 133.73, 133.04, 129.95,129.64, 129.15, 128.75, 128.63, 128.36, 80.00, 73.81, 73.72, 60.40,51.46, 31.90, 29.67, 29.65, 29.63, 29.59, 29.53, 29.51, 29.34, 28.30,25.72, 22.67, 14.11. HRMS (ESI-TOF) for C₃₇H₅₅NO₇Na⁺ [M+Na]⁺ calcd648.3871, found 648.3866.

Synthesis of Compound E:

To a stirred solution of D (1.5 g, 2.5 mmol), d (1.8 g, 3.0 mmol) andAW300 (2.0 g) in Et₂O-THF (7:1, 34 mL) was cooled to −40° C. and addedBF₃.OEt₂ (0.63 mL, 5 mmol). The solution was stirred for 4 h at ambienttemperature, and warmed to room temperature. The solution was filtered,added sat. NaHCO₃ aq. and extracted with AcOEt. The organic layer wasdried with MgSO₄ and evaporated to dryness. The residue was purified byflash column chromatography on silica gel (Hex.:AcOEt 5:1) to give acoupled product (980 mg, 38%) as a colorless oil.

To a stirred solution of this compound (980 mg, 0.95 mmol) in EtOH (30mL) was added 10% Pd—C (490 mg) and stirred vigorously under H₂atmosphere overnight. The solution was filtered and concentrated todryness. The residue and DMAP (cat.) were dissolved with pyridine (10mL) and Ac₂O (10 mL), stirred at room temperature overnight. Thesolution was concentrated, dissolved with AcOEt, washed with brine andconcentrated to dryness. The residue was purified by flash columnchromatography on silica gel (Hex.:AcOEt 2:1) to give E (790 mg, 87%) asa colorless oil. ¹H NMR (600 MHz, CDCl₃) δ: 8.00 (2H, d, J=7.3 Hz), 7.93(2H, d, J=7.5 Hz), 7.60 (1H, t, J=7.4 Hz), 7.52 (1H, t, J=7.4 Hz), 7.47(2H, t, J=7.8 Hz), 7.37 (2H, t, J=7.7 Hz), 5.66 (1H, dd, J=2.3 Hz, 9.6Hz), 5.41-5.48 (2H, m), 5.29-5.33 (2H, m), 5.16 (1H, dd, J=3.6 Hz, 10.9Hz), 4.82 (1H, d, J=3.5 Hz), 4.26 (1H, t, J=9.8 Hz), 4.17 (1H, t, J=6.7Hz), 4.06 (1H, dd, J=6.1 Hz, 11.3 Hz), 4.00 (1H, dd, J=7.1 Hz, 11.3 Hz),3.78 (1H, dd, J=2.4 Hz, 10.7 Hz), 3.49 (1H, dd, J=2.4 Hz, 10.7 Hz), 2.10(3H, s), 2.02 (3H, s), 1.99 (3H, s), 1.98 (3H, s), 1.90-1.99 (2H, m),1.52 (9H, s), 1.20-1.37 (24H, m), 0.88 (3H, t, J=7.0 Hz). ¹³C NMR (150MHz, CDCl₃) □: 170.58, 170.28, 170.11, 170.01, 166.11, 164.97, 155.11,133.34, 132.90, 129.92, 129.70, 129.56, 129.37, 129.52, 128.22, 97.35,80.28, 73.88, 71.81, 67.84, 67.70, 67.60, 66.97, 66.50, 61.67, 49.94,31.83, 29.60, 29.58, 29.56, 29.51, 29.43, 29.27, 29.20, 28.27, 28.13,25.63, 22.60, 20.60, 20.58, 20.52, 20.49, 14.05. HRMS (ESI-TOF) forC₅₁H₇₃NO₁₆Na⁺ [M+Na]⁺ calcd 978.4821, found 978.4814.

Alternatively, to a stirred solution of D (1.0 g, 1.6 mmol) d (1.4 g,2.4 mmol) and AW300 (2.0 g) in Et2O-THF (2:1, 30 mL) was cooled to −30°C. and added TMSOTf (18 μL, 0.081 mmol). The solution was stirred for 1h at ambient temperature, and d (0.9 g, 1.5 mmol) and TMSOTf (20 μL,0.090 mmol) was added. The solution was stirred an additional 1 h andthen warmed to −10° C. The solution was added triethylamine (2 mL), thenfiltered, added sat. NaHCO₃ aq. and extracted with AcOEt. The organiclayer was dried with MgSO4 and evaporated to dryness. The residue waspurified by flash column chromatography on silica gel (Hex.:AcOEt 5:1)to give a coupled product (1.3 g, 77%) as a colorless oil.

To a stirred solution of this compound (1.3 g, 1.2 mmol) in AcOEt (30mL) and EtOH (30 mL) was added 10% Pd—C (600 mg) and stirred vigorouslyunder H₂ atmosphere overnight. The solution was filtered andconcentrated to dryness. The residue and DMAP (cat.) were dissolved inpyridine (10 mL) and Ac₂O (10 mL), stirred at room temperatureovernight. The solution was concentrated, dissolved with AcOEt, washedwith brine and concentrated to dryness. The residue was purified byflash column chromatography on silica gel (Hex.:AcOEt 2:1) to give 42(1.1 g, 93%) as a colorless oil. ¹H NMR (600 MHz, CDCl₃) δ: 8.00 (2H, d,J=7.3 Hz), 7.93 (2H, d, J=7.5 Hz), 7.60 (1H, t, J=7.4 Hz), 7.52 (1H, t,J=7.4 Hz), 7.47 (2H, t, J=7.8 Hz), 7.37 (2H, t, J=7.7 Hz), 5.66 (1H, dd,J=2.3 Hz, 9.6 Hz), 5.41-5.48 (2H, m), 5.29-5.33 (2H, m), 5.16 (1H, dd,J=3.6 Hz, 10.9 Hz), 4.82 (1H, d, J=3.5 Hz), 4.26 (1H, t, J=9.8 Hz), 4.17(1H, t, J=6.7 Hz), 4.06 (1H, dd, J=6.1 Hz, 11.3 Hz), 4.00 (1H, dd, J=7.1Hz, 11.3 Hz), 3.78 (1H, dd, J=2.4 Hz, 10.7 Hz), 3.49 (1H, dd, J=2.4 Hz,10.7 Hz), 2.10 (3H, s), 2.02 (3H, s), 1.99 (3H, s), 1.98 (3H, s),1.90-1.99 (2H, m), 1.52 (9H, s), 1.20-1.37 (24H, m), 0.88 (3H, t, J=7.0Hz). ¹³C NMR (150 MHz, CDCl₃) δ: 170.58, 170.28, 170.11, 170.01, 166.11,164.97, 155.11, 133.34, 132.90, 129.92, 129.70, 129.56, 129.37, 129.52,128.22, 97.35, 80.28, 73.88, 71.81, 67.84, 67.70, 67.60, 66.97, 66.50,61.67, 49.94, 31.83, 29.60, 29.58, 29.56, 29.51, 29.43, 29.27, 29.20,28.27, 28.13, 25.63, 22.60, 20.60, 20.58, 20.52, 20.49, 14.05. HRMS(ESI-TOF) for C₅₁H₇₃NO₁₆Na⁺ [M+Na]⁺ calcd 978.4821, found 978.4814.

General procedure of synthesis of fatty acid chain analogues was asfollows:

To a stirred solution of E (240 mg, 0.25 mmol) in CH₂Cl₂ (2.4 mL) wasadded TFA (2.4 mL) at 0° C. and stirred for 2 h at ambient temperature.The solution was evaporated to dryness and co-evaporated with toluene 3times to give deprotected amine. This compound was dissolved in CH₂Cl₂and used for the next reaction without further purification.

To the deprotected amine (0.021 mmol) in CH₂Cl₂ (1.0 mL) was addedR—COOH (0.031 mmol), HBTu (12 mg, 0.031 mmol) and NMM (31 mg, 0.3 mmol)and stirred at room temperature overnight. The solution was purified byflash column chromatography on silica gel (Hex.:AcOEt 2:1) to give thecoupled product as a amorphic solids.

These compounds were dissolved in MeOH (2.0 mL) and 0.5 mol/L NaOMe inMeOH (4 drops) was added. The solution was stirred overnight at roomtemperature and evaporated to dryness. The residues were purified byflash column chromatography on silica gel (CHCl₃:MeOH 10:1) to giveR_(1,2).

Compound R_(1,2) were synthesized in a manner similar to that describedabove.

Intermediate of R₁: Yield 28 mg (65%). ¹H NMR (600 MHz, CDCl₃) □: 8.00(2H, dd, J=1.2 Hz, 8.2 Hz), 7.91 (2H, dd, J=1.2 Hz, 8.3 Hz), 7.61 (1H,t, J=7.4 Hz), 7.53 (1H, t, J=7.4 Hz), 7.47 (2H, t, J=7.8 Hz), 7.38 (2H,t, J=7.8 Hz), 7.25-7.28 (2H, m), 7.15-7.20 (3H, m), 6.57 (1H, d, J=9.7Hz), 5.69 (1H, dd, J=2.4 Hz, 9.9 Hz), 5.43 (1H, d, J=3.3 Hz), 5.33 (1H,dd, J=3.4 Hz, 10.9 Hz), 5.29-5.32 (1H, m), 5.15 (1H, dd, J=3.6 Hz, 10.9Hz), 4.81 (1H, d, J=3.6 Hz), 4.62 (1H, tt, J=2.5 Hz, 9.9 Hz), 4.11 (1H,dd, J=6.6 Hz, 13.3 Hz), 4.05 (1H, dd, J=6.0 Hz, 11.0 Hz), 3.96 (1H, dd,J=7.0 Hz, 11.3 Hz), 3.73 (1H, dd, J=2.8 Hz, 10.9 Hz), 3.49 (1H, dd,J=2.4 Hz, 10.9 Hz), 2.64 (2H, t, J=7.8 Hz), 2.35 (2H, t, J=7.7 Hz), 2.10(3H, s), 1.994 (3H, s), 1.986 (3H, s), 1.94 (3H, s), 1.89-1.93 (2H, m),1.66-1.78 (4H, m), 1.42-1.48 (2H, m), 1.18-1.35 (24H, m), 0.87 (3H, t,J=7.1 Hz). ¹³C NMR (150 MHz, CDCl₃) □: 172.90, 170.59, 170.39, 170.21,170.15, 166.50, 165.03, 142.53, 133.49, 133.07, 129.87, 129.77, 129.62,129.30, 128.62, 128.38, 128.32, 128.24, 125.62, 97.29, 74.14, 71.45,67.90, 67.53, 67.32, 67.10, 66.68, 61.74, 48.18, 36.65, 35.76, 31.90,31.22, 29.65, 29.63, 29.60, 29.56, 29.53, 29.34, 29.32, 28.99, 27.86,25.69, 25.57, 22.67, 20.67, 20.62, 20.58, 20.50, 14.11. HRMS (ESI-TOF)for C₃₆H₆₄NO₉ ⁺ [M+H]⁺ calcd 1030.5522, found 1030.5507.

R₁: Yield 14 mg (79%). ¹H NMR (500 MHz, CDCl₃-MeOH 4:1) □: 7.25-7.29(2H, m), 7.15-7.19 (3H, m), 4.90 (1H, d, J=3.9 Hz), 4.17-4.21 (1H, m),3.94 (1H, d, J=3.2 Hz), 3.87 (1H, d, J=4.7 Hz), 3.67-3.81 (6H, m),3.51-3.56 (2H, m), 2.61 (2H, t, J=7.8 Hz), 2.20 (2H, t, J=7.6 Hz),1.44-1.70 (6H, m), 1.21-1.41 (26H, m), 0.88 (3H, t, J=7.0 Hz). ¹³C NMR(125 MHz, CDCl₃-MeOH 4:1) □: 174.08, 142.25, 128.10, 128.01, 125.42,99.49, 74.64, 71.86, 70.49, 70.04, 69.53, 68.70, 67.27, 61.69, 50.17,36.14, 35.46, 32.49, 31.67, 30.93, 29.54, 29.49, 29.46, 29.40, 29.11,29.00, 28.67, 25.60, 25.40, 22.42, 13.76. HRMS (ESI-TOF) for C₃₆H₆₄NO₉ ⁺[M+H]⁺ calcd 654.4575, found 654.4568.

Intermediate of R₂: Yield 28 mg (65%). ¹H NMR (600 MHz, CDCl₃) δ: 7.99(2H, d, J=7.7 Hz), 7.91 (2H, d, J=7.9 Hz), 7.61 (1H, t, J=7.4 Hz), 7.53(1H, t, J=7.4 Hz), 7.47 (2H, t, J=7.7 Hz), 7.37 (2H, t, J=7.7 Hz),7.24-7.28 (2H, m), 7.14-7.18 (3H, m), 6.62 (1H, d, J=9.8 Hz), 5.71 (1H,dd, J=2.3 Hz, 9.9 Hz), 5.43 (1H, d, J=3.2 Hz), 5.35 (1H, dd, J=3.3 Hz,10.9 Hz), 5.29-5.31 (1H, m), 5.15 (1H, dd, J=3.6 Hz, 10.9 Hz), 4.81 (1H,d, J=3.6 Hz), 4.59-4.64 (1H, m), 4.09-4.12 (1H, m), 4.06 (1H, dd, J=5.9Hz, 11.2 Hz), 3.97 (1H, dd, J=7.0 Hz, 11.3 Hz), 3.73 (1H, dd, J=2.7 Hz,10.8 Hz), 3.48 (1H, dd, J=2.2 Hz, 10.9 Hz), 2.60 (2H, t, J=7.8 Hz), 2.35(2H, t, J=7.7 Hz), 2.10 (3H, s), 2.005 (3H, s), 1.996 (3H, s), 1.94 (3H,s), 1.89-1.93 (2H, m), 1.59-1.76 (4H, m), 1.19-1.43 (30H, m), 0.87 (3H,t, J=7.0 Hz). ¹³C NMR (150 MHz, CDCl₃) □: 172.99, 170.60, 170.38, 17023,170.16, 166.52, 165.03, 142.80, 133.47, 133.07, 129.87, 129.76, 129.61,129.30, 128.61, 128.36, 128.31, 128.18, 125.52, 97.24, 74.16, 71.35,67.93, 67.52, 67.29, 67.12, 66.70, 61.77, 48.15, 36.71, 35.92, 31.89,31.47, 29.66, 29.63, 29.60, 29.56, 29.53, 29.34, 29.30, 29.26, 29.23,29.19, 27.80, 25.72, 25.67, 22.66, 20.67, 20.63, 20.58, 20.49, 14.11.HRMS (ESI-TOF) for C₆₀H₈₄NO₁₅ ⁺ [M+H]⁺ calcd 1058.5835, found 1058.5819.

R₂: Yield 14 mg (79%). ¹H NMR (500 MHz, CDCl₃-MeOH 4:1) δ: 7.25-7.29(2H, m), 7.15-7.18 (3H, m), 4.91 (1H, d, J=3.8 Hz), 4.17-4.22 (1H, m),3.94 (1H, d, J=3.2 Hz), 3.87 (1H, d, J=4.7 Hz), 3.67-3.81 (6H, m),3.51-3.56 (2H, m), 2.60 (2H, t, J=7.7 Hz), 2.19 (2H, t, J=7.7 Hz),1.49-1.70 (6H, m), 1.20-1.41 (30H, m), 0.88 (3H, t, J=7.0 Hz). ¹³C NMR(125 MHz, CDCl₃-MeOH 4:1) □: 174.18, 142.54, 128.11, 127.97, 125.34,99.49, 74.64, 71.86, 70.48, 70.05, 69.54, 68.71, 67.28, 61.71, 50.17,36.28, 36.23, 35.67, 32.47, 31.67, 31.24, 29.54, 29.49, 29.47, 29.41,29.11, 29.04, 29.01, 28.92, 25.60, 25.57, 22.42, 13.76. HRMS (ESI-TOF)for C₃₈H₆₈NO₉ ⁺ [M+H]⁺ calcd 682.4888, found 682.4880.

In general, the phytosphingosine skeleton was constructed bymodification of a method described by Savage and co-workers [R. D. Goff,et al. J. Am. Chem. Soc., 2004, 126, 13602-13603] Garner's aldehyde Awas coupled with a Wittig reagent prepared from phosphonium bromide Baccording to Berova's method [O. Shirota, et al., Tetrahedron, 1999, 55,13643-13658] to give cis olefin B in 66% yield. Treatment of olefin Bwith osmium tetroxide gave a corresponding diol C and its undesiredisomer. The two hydroxyl groups of diol C were protected with benzoylgroups, and then the isopropylidene group was removed by the successivetreatment of TFA, followed by Boc anhydride protection to affordphytosphingosine acceptor D in 67% yield over 3 steps.

Glycosylation of phytosphingosine acceptor D and donor d in the presenceof BF₃.OEt₂ gave a predominantly α-configured product. Hydrogenation wasavoided as the final deprotection step to ensure accessibility to a morediverse set of compounds. The galactose protecting groups were removedand then protected with acetates to furnish the key intermediate E in33% over 3 steps.

Compound E was deprotected with TFA to give the deprotected amine. Avariety of fatty acyl chain analogues were then couples to the amine toform R after removal of the acetyl groups.

A synthetic scheme for Phytosphingosine Analogues is provided in scheme2 below:

-   -   wherein R is selected from Table 2 below to provide the        corresponding compound.

TABLE 2 Compound No. R = 112

113

Synthesis of phytosphingosine chain analogues: The synthesis ofphytosphingosine chain analogues PC6Ph, PC8Ph, PC11Ph and PC13Ph aresummarized in scheme 2. The synthetic route is essentially similar tothe one reported in J. Am. Chem. Soc. 2006, 128, 9022-9023, supportinginformation.

Characterization Data:

Compound 112 (lot. MFJ3-017-1): ¹H NMR (500 MHz, CDCl₃-MeOH 4:1) δ: 7.26(m, 2H), 7.23-7.19 (m, 2H), 7.18-7.14 (m, 1H), 4.90 (d, J=3.9 Hz, 1H),4.24-4.19 (m, 1H), 3.86 (dd, J=10.8, 5.2 Hz, 1H), 3.82-3.62 (m, 7H),3.58-3.53 (m, 2H), 2.92-2.84 (m, 1H), 2.67 (ddd, J=13.7, 9.3, 7.5 Hz,1H), 2.16 (m, 2H), 2.06-1.98 (m, 1H), 1.74-1.65 (m, 1H), 1.62-1.53 (m,2H), 1.33-1.19 (m, 44H), 0.88 (t, J=7.0 Hz, 3H). ¹³C NMR (125 MHz,CDCl₃-MeOH 4:1) δ: 174.06, 141.93, 128.25, 128.01, 125.43, 99.48, 74.60,70.75, 70.44, 69.99, 69.52, 68.66, 67.03, 61.69, 50.15, 50.06, 36.27,34.13, 31.67, 31.59, 29.43, 29.31, 29.15, 29.09, 25.55, 22.41, 17.60,13.76. HRMS (ESI-TOF) for C₄₄H₈₀NO₉ ⁺ [M+H]⁺ calcd 766.5827, found766.5813.

Compound 113 (lot. MFJ3-018-1): ¹H NMR (400 MHz, CDCl₃-MeOH 4:1) δ: 7.26(m, 2H), 7.19-7.13 (m, 3H), 4.91 (d, J=3.8 Hz, 1H), 4.20 (q, J=4.4 Hz,1H), 3.95-3.85 (m, 2H), 3.83-3.61 (m, 6H), 3.59-3.50 (m, 2H), 2.63 (t,J=7.5 Hz, 2H), 2.20 (t, J=7.5 Hz, 2H), 1.78-1.54 (m, 6H), 1.47-1.17 (m,46H), 0.89 (t, J=6.9 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃-MeOH 4:1) δ:174.16, 142.27, 127.91, 127.77, 125.14, 99.33, 74.28, 71.38, 70.42,69.86, 69.33, 68.51, 66.84, 61.40, 50.02, 36.04, 35.52, 31.93, 31.51,31.21, 29.26, 29.14, 28.99, 28.94, 25.47, 25.08, 22.25, 13.51. HRMS(ESI-TOF) for C₄₆H₈₄NO₉ ⁺ [M+H]⁺ calcd 794.6140, found 794.6129.

Example 8 Recognition of Glycolipids by Murine NKT Cell Lines Results inIL-2 Secretion Materials and Methods

Glycolipids

The compound KRN 7000 was purchased (Kirin, Japan). The remainingcompounds were synthesized as described hereinabove.

1.2 Hybridoma Assay

CD1d reactive T cell hybridomas with an invariant Va14i T cell antigenreceptor α chain were used, as described (Sidobre, S., et al. (2004)Proc. Natl. Acad. Sci. USA 101, 12254-12259). T cell hybridomas werestimulated with 0.0001-1 μg/ml of the indicated glycolipids added toCD1d transfected A20 B lymphoma cells, as described (Elewaut, D., et al.(2003) J. Exp. Med. 198, 1133-1146). As a measure of T cell activation,IL-2 and IFN-γ release into the tissue culture medium was measured after16 hours culture by an ELISA assay.

In Vitro Cytokine Secretion Assay Using Human NKT Cell Lines

IL-2 secretion by the Vα24i human NKT cell line was determined by ELISA(BD Pharmingen, San Diego, Calif.) after culture for 16 hours. For theseassays, 1×10⁵ Vα24i human NKT cells were co-cultured with 4×10⁵irradiated, immature CD14⁺ dendritic cells, in the presence of theglycolipid compounds at 10 μg/ml in a 96-well flat-bottom plate [Wu etal. PNAS 2005 102: 1351].

Results

In order to test whether glycolipids with modifications the lipid moietyof α-GalCer affected the immunogenicity of the compound, a series ofglycolipids with varied modifications of this region were synthesizedand assayed.

Mouse Vα14i NKT cells immortalized by cell fusion provided a convenientmethod for assaying the ability of the synthetic glycolipids to activateT cells. As shown in FIG. 12, a number of the compounds (60, 61, 62, 64,65, 74, 77) stimulated significant IL-2 release from the hybridomas whenused at 1 μg/ml, however KRN7000 (α-GalCer) appeared to stimulate thegreatest amount of IL-2 release.

Another series of compounds were evaluated for their ability tostimulate IL-2 release, when provided at various concentrations (FIG.13). In this case, several of these compounds stimulated greater IL-2release, as compared to KRN7000 (α-GalCer), in particular, compoundswith a terminal phenyl substituent.

The simplest benzoyl analog 58 showed only slight activity. Introductionof either electron donating groups (68; 4-OCH3, 69; 4-CH3) orwithdrawing groups (70; 4-Cl, 59; 4-CF3) onto the benzene ring increasedtheir activity. The other benzoyl analogues, 4-Pyridyl 80, 3-pyridyl 71,indole analog 81 and biphenyl analogues 72, 63 and 80 also exhibitedsimilar trends. However, their activities still remained about half ofα-GalCer.

Benzyl analogues 60, 61, 69, 73 and 74 showed improved IL-2 productioncompared with the benzoyl analogues. Of these compounds, smalleraromatic groups such as 60, 61 and 69 showed better activity than thatof analogues 73, 74 bearing larger aromatic groups. The activities ofthiophene analog 69 and benzene analog 60 were comparable.

Phenylethylene analogues 62, 77, 87 and 88 demonstrated comparable oreven more potent IL-2 production compared with the benzyl analogues. The4-CF3 analog 87 and 4-isobutyl analog 82 possessed slightly betteractivities compared with the 4-OCH3 analog 62 and 4-F analog 88.Substitution of the phenyl group with the 3-pyridyl group 86 diminishedIL-2 production dramatically, which contradicted the trends of benzoylanalogues (58 and 71). In addition, the introduction of a trans-doublebond as a spacer group significantly reduced IL-2 production comparedwith the saturated analog (75 and 77). The biphenyl analog 93 alsoshowed a decreased activity. Introduction of a basic functional group,such as piperidinylethyl analog 91 demonstrated a significant reductionin cytokine production. This result may be because of repulsion betweenthe basic amine moiety and the hydrophobic residue in the bindingpocket. The 4-Fluorophenoxymethyl analog 65 gave a similar activity asthe corresponding carbon analog 88. On the other hand, the 2,6-dimethylsubstituted analog 66 exhibited a reduced activity, suggesting that thebinding pocket was not large enough to accept bulky substituents.Similar results were observed in compounds 72, 89 and 90, bearing bulkysubstituent such as 4-biphenylmethyl, 2,2-diphenylmethyl and9-fluorenyl, respectively.

Further extension of spacer chain length gave best results under theseconditions. The activity of 3-phenylpropyl analog 82 was moderate.However, the 5-phenylpentyl 83, 7-phenylheptyl 84 and 10-phenyloctyl 85all showed a significant increase of IL-2 production. Compounds 83-85were much more potent than α-GalCer.

Example 9 Recognition of Glycolipids by Human NKT Cell Lines Results inNKT Cell IFN-γ and IL-4 Secretion Materials and Methods

Generation of Vα24i Human NKT Cell Line

Human NKT cell lines, expressing the Vα24i T cell receptor as well asCD161, were generated as follows: Anti-CD161 monoclonal antibodies, andanti-CD14 monoclonal antibodies, each coupled to magnetic beads(Miltenyi biotec, Auburn, Calif.), were used sequentially to isolateCD161⁺ cells and CD14⁺ cells from leukopaks. Immature dendritic cellswere generated from the CD14⁺ cells after a two-day incubation in thepresence of 300 U/ml GM-CSF (R&D systems, Minneapolis, Minn.) and 100U/ml IL-4 (R&D systems, Minneapolis, Minn.). Following irradiation with2000 rads, the immature dendritic cells were co-cultured with syngeneicCD161⁺ cells in the presence of 100-0.1 ng/ml ofalpha-galactosylceramide and 10 IU/ml of IL-2 (Invitrogen, CarlsbadCalif.) for 10 to 14 days. After stimulating the CD161⁺ cells a secondtime with alpha-galactosylceramide-pulsed, irradiated immature dendriticcells, NKT cell lines were shown by flow cytometry to express bothCD161⁺ and V 24i TCR (99% purity).

In some cases, Hela cells were transfected with a human CD1d construct[Xing et al. 2005. Bioorg Med Chem 13: 2907], and were used to presentthe glycolipids, via pulsing with the respective compounds at theindicated concentration, to NKT lines.

IFN-γ secretion by the Vα24i human NKT cell line was determined by ELISA(BD Pharmingen, San Diego, Calif.) after culture for 16 hours. For theseassays, 1×10⁵ Vα24i human NKT cells were co-cultured with 4×10⁵irradiated, immature CD14⁺ dendritic cells, in the presence of theglycolipid compounds at 10 μg/ml in a 96-well flat-bottom plate.

Results

IFN-γ secretion from a Vα24i NKT cell line were assessed, afterstimulation with irradiated, syngeneic CD14⁺ immature dendritic cells inthe presence of 10, 1 and 0.1 μg/ml of the respective glycolipids and 10IU/ml of IL-2 (FIGS. 14, 15 and 16).

Stimulation of the NKT cell line by many of the glycolipid compoundsresulted in significant IFN-γ secretion, when compared to the negativecontrol, with some specific compounds providing greater greater IFN-γsecretion as compared to α-GalCer.

As illustrated in FIGS. 17 and 18, additional glycolipid compounds wereprepared and evaluated for interferon-γ secretion by human NKT cells inresponse to glycolipid presentation by CD14⁺ DCs, as compared toα-GalCer, at a concentration of 100-0.1 ng/mL. Compounds 83, 84 and 85in these figures consistently stimulating greater IFN-γ secretion, atall doses evaluated, as compared to KRN, and other compounds.

Hela cells expressing human CD1d were also effective in presenting theglycolipids to human NKT cells, with similar profiles in terms ofstimulating NKT cell IFN-γ secretion (FIG. 19).

Compounds effective in stimulating IFN-g secretion were also found tostimulate IL-4 secretion (FIG. 20).

The longer alkyl chain analogues 83-85 were more potent toward bothIFN-γ and IL-4 production than the shorter alkyl chain analogues. The7-phenylheptyl analog 83 exhibited a high ratio of IFN-γ/IL-4 activityand was the best among these compounds. However, compounds 73 and 77 aremore selective for IL-4 production while 82 is specific for IFN-γproduction.

Example 10 Possible Structural Basis for Glycolipid Recognition

Recent glycolipid-CD1d protein crystal structures revealed the existenceof various aromatic residues, Tyr73, Phe114, Phe70 and Tip114, whichmight be able to interact with the phenyl group of the present compoundscomprising fatty acyl chain analogues. According to these crystalstructures, the benzoyl analogues 8-14, which have no spacer chain,seemed to be too short to interact with these aromatic residues. Tofurther investigate the interactions between the phenyl analogues andhuman CD1d, Autodock 3.0 [G. M. Morris, et al., J. Comp. Chem., 1998,19, 1639-1662] was utilized to model the binding of these compounds inthe hCD1d hydrophobic groove (FIG. 21). Compounds 40-42 wereindividually docked and their results did not vary significantly fromthe crystal structure of α-GalCer bound to hCD1d [M. Koch, et al., Nat.Immunol., 2005, 6, 819-826]. In each case, the phytosphingosine tailextended into the F′ pocket and the A′ pocket was occupied by the fattyacyl chain with the galactose headgroup presented in nearly all the sameconfiguration. However, introduction of a terminal phenyl group in theα-GalCer analogues seemed to promote additional specific interactionsbetween compounds 40, 41 and the phenol ring of Tyr73 and between 42 andTrp40.

Biphenyl analogues 16-18 and cinnamoyl analogues 30, 32, lacked aflexible fatty acyl chain and may not have been able to extend into theA′ pocket deep enough to make any specific interactions. Extension ofthe spacer chain length, such as the benzyl analogues 19-22, thephenylethylene analogues 24-28, and the 4-fluorophenoxymethyl analog 34,allowed for tighter binding via π-π interaction, possibly with thearomatic side-chain of Tyr73. Further extension of the spacer chainlength, such as pentamethylene analog 83, heptamethylene analog 84 anddecamethylene analog 85, were more suitable for tighter binding. Theseresults suggest that the introduction of π-π interaction potentiatesIL-2 production, probably through the formation of a tighter ligand—CD1dprotein complex.

These fatty acyl chain analogues bearing aromatic groups seemed topossess more potent activity than the corresponding simple fatty acylchain analogues. Compounds 83-85 bearing 5, 7, 10 carbons spacer chain,respectively, demonstrated much more potent IL-2 production than that ofother groups, with α-GalCer bearing a C26 fatty acyl chain. Theseresults suggest that introduction of a terminal aromatic group on thefatty acyl chain causes an enhancement of the activity throughinteractions between the aromatic residues in the hydrophobic pocket ofCD1d protein and the lipid tail.

Example 11 In Vivo Mouse Assay

Wild-type C57BL/6 (B6) mice were administered intravenously with 1 μg ofeach glycolipid or with nothing, and serum samples were obtained at 2,6, 12, 24, 48, and 72 hours after injection for ELISA analyses of IL-4,IFN-γ, and IL-12 concentrations. The serum concentrations of IFN-γ andIL-4 were measured by way of a sandwich ELISA (e-bioscience, San Diego,Calif.). The serum concentrations of IL-12p70 were also measured by wayof a sandwich ELISA (PharMingen, San Diego, Calif.). Results areprovided in FIG. 22.

It will be appreciated by a person skilled in the art that the presentinvention is not limited by what has been particularly shown anddescribed hereinabove. Rather, the scope of the invention is defined bythe claims that follow:

1. A method for enhancing immunogenicity of a compound, composition, orvaccine in a subject, the method comprising steps: (a) administering tothe subject a compound, composition, or vaccine designed to stimulate aprotective immune response, wherein the compound, composition, orvaccine, comprises an adjuvant, wherein the adjuvant comprises acompound represented by the structure of formula 1:

wherein, R═COOR₁ or CH₂OR₁; R₁═H or an alkyl group; R₂═H Or SO₃ ⁻; R₃═Hor OH; R₃′═H or OH; R₄ and R₄′ are independently chosen from (a) H, (b)alkyl; (c) alkenyl; and (d) oxaalkyl; R₅═OH, acetamido or a halogenatom; or a pharmaceutically acceptable salt thereof, wherein if R═CH₂OR₁where R₁ is H, then R₂═SO₃ ⁻, and wherein if R═CH₂OR₁, R₂═H, R₃ is OHand R₃′ is H, then R₅=acetamido or a halogen atom.
 2. A method forenhancing immunogenicity of a compound, composition, or vaccine in asubject, the method comprising steps: (a) administering to the subject acompound, composition, or vaccine designed to stimulate a protectiveimmune response, wherein the compound, composition, or vaccine comprisesan adjuvant, wherein the adjuvant comprises a compound represented bythe structure of formula (17):

wherein R₄ is selected from: (a) alkyl, (b) alkenyl, (c) alkylterminating in aryl, substituted aryl, heteroaryl or substitutedheteroaryl; and (d) alkenyl terminating in aryl, substituted aryl,heteroaryl or substituted heteroaryl, and R₆ is

X is an alkyl chain; and R₇ is chosen from halogen, H, phenyl, alkyl,alkoxy, nitro, and CF₃.
 3. The method according to claim 2, wherein R₄and X are saturated alkyl groups containing from 1 to 30 carbons.
 4. Themethod according to claim 3, wherein R₄ and X are saturated alkyl groupscontaining from 5 to 25 carbons.
 5. The method according to claim 4,wherein R₇ is H.
 6. The method according to claim 5, wherein thecompound is selected from the group consisting of:


7. The method according to claim 4, wherein R₇ is halogen.
 8. The methodaccording to claim 2, wherein the compound is selected from the groupconsisting of:


9. The method according to claim 4, wherein R₇ is alkoxy.
 10. The methodaccording to claim 9, wherein the compound is selected from the groupconsisting of:


11. The method according to claim 4, wherein R₇ is CF₃.
 12. The methodaccording to claim 11, wherein the compound is selected from the groupconsisting of:


13. The method according to claim 4, wherein R₇ is phenyl.
 14. Themethod according to claim 13, wherein the compound is selected from thegroup consisting of:


15. The method according to claim 2, wherein R₄ is

X is an alkyl chain; and R₇ is chosen from halogen, H, phenyl, alkyl,alkoxy, nitro, and CF₃.
 16. The method according to claim 15, wherein R₆and X are saturated alkyl groups containing from 1 to 30 carbons. 17.The method according to claim 16, wherein R₆ and X are substituted alkylgroups containing from 2 to 10 carbons.
 18. The method according toclaim 17, wherein R₇ is H.
 19. The method according to claim 18, whereinthe compound is selected from the group consisting of: